Selected reaction monitoring assays

ABSTRACT

Provided herein are methods for developing selected reaction monitoring mass spectrometry (LC-SRM-MS) assays.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/738,146, filed Jun. 12, 2015, which is a continuation of U.S.application Ser. No. 13/725,098, filed Dec. 21, 2012, now U.S. Pat. No.9,091,651, which claims priority and benefit of U.S. ProvisionalApplication No. 61/578,718, filed Dec. 21, 2011, and U.S. ProvisionalApplication No. 61/614,818, filed Mar. 23, 2012, the contents of each ofwhich are incorporated herein by reference in their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “IDIA-004_C01US_ST25.txt”, which wascreated on Jun. 11, 2015 and is 395,000 bytes in size, are herebyincorporated by reference in their entireties.

BACKGROUND

Liquid Chromatography Selected Reaction Monitoring Mass Spectrometry(LC-SRM-MS) has emerged as an alternative technology to immunoassays forquantification of target proteins in biological samples. LC-SRM-MSmethods are highly desirable because LC-SRM-MS methods provide bothabsolute structural specificity for the target protein and relative orabsolute measurement of the target protein concentration when suitableinternal standards are utilized. In contrast to immunoassays, LC-SRM-MSdoes not involve the manufacturing of biologics. LC-SRM-MS proteinassays can be rapidly and inexpensively developed in contrast to thedevelopment of immunoassays. LC-SRM-MS are highly multiplexed, withsimultaneous assays for hundreds of proteins performed in a singlesample analysis. Using LC-SRM-MS in contrast to other proteomictechnologies allows for complex assays for the identification diagnosticproteins in complex diseases such as cancer, autoimmune, and metabolicdisease. In particular, the development of a highly multiplexedLC-SRM-MS assay that reproducibly identifies a specific set of proteinsrelevant to a clinical disease presents diagnostic advantages andefficiencies. To date, proteomic techniques have not enabled suchinventions to exist where hundreds of proteins can be accuratelyquantified within a single sample. The present invention providesaccurate measurement of hundreds of lung cancer associated proteinswithin a single sample using multiplexed techniques.

SUMMARY OF THE INVENTION

The present invention comprises a LC-SRM-MS assay for the measurementproteins in a single sample and in a single LC-SRM-MS assay. The assaywas optimized for protein quantification and minimal interference amongproteins in the assay. This LC-SRM-MS assay is novel because measurementof a large number of proteins in a single sample specifically associatedwith lung cancer has not been accomplished. Simultaneous measurement ofsuch a large number of proteins without interference among the proteinsrequires specific techniques to distinguish among the proteins. Thecurrent invention provides clinical utility as this assay was used fordevelopment of lung cancer diagnostic tests for the early detection oflung cancer, managing disease treatment, as well as testing for diseaserecurrence.

The object of the present invention is to provide improved methods forthe use of LC-SRM-MS in the development of assays. Accordingly, providedherein is a method for developing peptides and transitions for aplurality of at least 200 proteins for a single sample selected reactionmonitoring mass spectrometry (LC-SRM-MS) assay, including the steps ofproviding a set of 200 or more proteins; generating transitions for eachprotein; assessing LC-SRM-MS data by Mascot score; performing collisionenergy optimization on the transitions; selecting peptides withtransitions showing the greatest peak areas of their transitions;selecting a set of transitions for each peptide, wherein the transitionsfor each peptide have one of the four most intense b or y transitionions; the transitions for each peptide have m/z values of at least 30m/z above or below those of the precursor ion; the transitions for eachpeptide do not interfere with transitions from other peptides; and thetransitions represent transitions due to breakage of peptide bond atdifferent sites of the protein.

In one embodiment of the method, each selected peptide in the set ofpeptides has a monoisotopic mass of 700-5000 Da; and does not contain acysteine or a methionine; or may contain cysteine or methionine. Inanother embodiment, the transitions for each peptide have one of thefour most intense b or y transition ions; have m/z values of at least 30m/z above or below those of a precursor ion; do not interfere withtransitions from other peptides; and represent transitions due tobreakage of peptide bond at different sites of the protein.

In another embodiment of the method, the peptides do not include anypeptide that is bounded by KK, KR, RK or RR (either upstream ordownstream) in the corresponding protein sequence. Specifically, theamino acid is charged at pH 7.0. These amino acids include arginine andlysine. In another embodiment, each peptide of said set of peptides isunique to the corresponding protein. In yet another embodiment, thepeptides do not include peptides which were observed inpost-translational modified forms. In still another embodiment, each setof peptides is prioritized according to one or more of the followingordered set of criteria: unique peptides first, then non-unique;peptides with no observed post-translational modifications first, thenthose observed with post-translational modifications; peptides withinthe mass range 800-3500 Da first, then those outside of 800-3500 Da; andsorted by decreasing number of variant residues. In certain embodiments,The peptides are unique in that they only appear once among the peptidesrun in a single assay.

In one embodiment, each set of peptides is prioritized according to allof the ordered set of criteria. In another embodiment, each prioritizedset of peptides contains 1-5 peptides.

In certain embodiments of the preceding methods, the two best peptidesper protein and the two best transitions per peptide are selected basedon experimental data resulting from LC-SRM-MS analysis of one or more ofthe following experimental samples: a biological disease sample, abiological control sample, and a mixture of synthetic peptides ofinterest. In a particular embodiment, the biological disease andbiological control samples are processed using an immunodepletion methodprior to LC-SRM-MS analysis. In another embodiment, the experimentalsamples contain internal standard peptides. In yet another embodiment,the LC-SRM-MS analysis method specifies a maximum of 7000 transitions,including transitions of the internal standard peptides and transitions.In other embodiments the method specifies a maximum of between1000-7000, 2000-6000, 3000-5000 and about 3500 transitions.

In one embodiment of the method, the top two transitions per peptide areselected according to one or more of the following criteria thetransitions exhibit the largest peak areas measured in either of the twobiological experimental samples; the transitions are not interfered withby other ions; the transitions do not exhibit an elution profile thatvisually differs from those of other transitions of the same peptide; orthe transitions are not beyond the detection limit of both of the twobiological experimental samples.

In another embodiment of the method, the top two peptides per proteinare selected according to one or more of the following criteria: one ormore peptides exhibit two transitions and represent the largest combinedpeak areas of the two transitions; or one or more peptides exhibit onetransition and represent the largest combined peak areas of the twotransitions.

In another aspect, provided herein is an assay developed according tothe foregoing method, and embodiments thereof.

In yet another aspect provided herein is the use of an assay developedaccording to the foregoing method, and embodiments thereof, to detect aplurality of at least 200 proteins in a single biological sample.

In another aspect, provided herein is an assay developed according tothe foregoing method, and embodiments thereof.

The disclosure provides a composition comprising at least fivetransition ions selected from the listing of transition ions in Table 2.In one embodiment of the assay each transition ion independentlycorresponds to a unique protein. The five transition ions correspondedto proteins selected from the group consisting of LRP1, BGH3, COIA1,TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1.(see: U.S. application Ser. No. 13/306,823 PCT/US11/62461). Thecomposition can further include an additional five transition ionsselected from the listing of transition ions in Table 2. The additionalfive transition ions can corresponded to the proteins APOE, BASP1, CD14,FOXA2 and HSPB1.

The disclosure provides a composition comprising at least five syntheticpeptides selected from the listing of peptides and proteins in Table 2.In one embodiment, each peptide can independently correspond to a uniqueprotein. At least one of the peptides was isotopically labeled. Theamount of each of the at least five synthetic peptides is known. Inanother embodiment, the composition included one or more polar solvents.The five synthetic peptides can correspond to the proteins LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, andGSLG1. The composition can also include an additional five syntheticpeptides selected from the listing of peptides and proteins in Table 2.The additional five synthetic peptides can correspond to the proteinsAPOE, BASP1, CD14, FOXA2 and HSPB1.

The disclosure provides a use of a composition, as described above, forthe development of an assay to detect a disease, disorder or conditionin a mammal.

The disclosure provides a method comprising analyzing a composition, asdescribed above, using mass spectrometry. The method can use selectedreaction monitoring mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts candidate protein cohort by source. 217 tissue proteinswere identified using proteomics analysis. 319 proteins were identifiedby review of the literature. Between the two sources, there was anoverlap of 148 proteins.

FIG. 2 is a bar diagram showing Pearson correlations for peptides fromthe same peptide, from the same protein and from different proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to methods for developing peptides andtransitions for a single sample selected reaction monitoring massspectrometry (LC-SRM-MS) assay, generally comprising the steps ofproviding a set of proteins; identifying representative proteolyticpeptides for each protein according to a set of criteria; identifyingrepresentative transitions for each peptide according to another set ofcriteria; and selecting the optimum peptides per protein and the optimumtransitions per peptide.

Selected reaction monitoring mass spectrometry is capable of highlysensitive and accurate protein quantification based on thequantification of proteolytic peptides. In terms of clinical utility,mass spectrometry-based assays are often compared to immunoassays (e.g.,Enzyme-Linked Immunosorbent Assay, or ELISA), which have the ability toquantify specific analytes in large sample sets (e.g., 96 or 384 samplesin parallel microtitre plate-based format). Until recently, massspectrometry-based protein assays were not able to match these samplesizes or quantitative accuracy. Considerable time and expense isrequired to generate and characterize antibodies required forimmunoassays. Increasingly efficient LC-SRM-MS assays, therefore, maysurpass immunoassays such as ELISA in the rapid development ofclinically useful, multiplexed protein assays.

LC-SRM-MS is a highly selective method of tandem mass spectrometry whichhas the potential to effectively filter out all molecules andcontaminants except the desired analyte(s). This is particularlybeneficial if the analysis sample is a complex mixture which maycomprise several isobaric species within a defined analytical window.LC-SRM-MS methods may utilize a triple quadrupole mass spectrometerwhich, as is known in the art, includes three quadrupole rod sets. Afirst stage of mass selection is performed in the first quadrupole rodset, and the selectively transmitted ions are fragmented in the secondquadrupole rod set. The resultant transition (product) ions are conveyedto the third quadrupole rod set, which performs a second stage of massselection. The product ions transmitted through the third quadrupole rodset are measured by a detector, which generates a signal representativeof the numbers of selectively transmitted product ions. The RF and DCpotentials applied to the first and third quadrupoles are tuned toselect (respectively) precursor and product ions that have m/z valueslying within narrow specified ranges. By specifying the appropriatetransitions (m/z values of precursor and product ions), a peptidecorresponding to a targeted protein may be measured with high degrees ofsensitivity and selectivity. Signal-to-noise ratio in LC-SRM-MS is oftensuperior to conventional tandem mass spectrometry (MS/MS) experimentsthat do not selectively target (filter) particular analytes but ratheraim to survey all analytes in the sample.

Accordingly, provided herein is a method for developing peptides andtransitions for a plurality of proteins for use in selected reactionmonitoring mass spectrometry (LC-SRM-MS) assay. In a preferredembodiment, the assay involves the analysis of a single samplecontaining all analytes of interest (e.g., a proteolytic digest ofplasma proteins). As to the selection of the protease(s) used, trypsin,which cleaves exclusively C-terminal to arginine and lysine residues, isa preferred choice to generate peptides because the masses of generatedpeptides are compatible with the detection ability of most massspectrometers (up to 2000 m/z), the number and average length ofgenerated peptides, and also the availability of efficient algorithmsfor the generation of databases of theoretical trypsin-generatedpeptides. High cleavage specificity, availability, and cost are otheradvantages of trypsin. Other suitable proteases will be known to thoseof skill in the art. Miscleavage is a factor for failure or ambiguousprotein identification. A miscleavage can be defined as partialenzymatic protein cleavages generating peptides with internal missedcleavage sites reflecting the allowed number of sites (targeted aminoacids) per peptide that were not cut. The presence of post-translationalmodifications (PTMs) is also a potential contributor to the problem ofmiscleavages.

LC-SRM-MS mass spectrometry involves the fragmentation of gas phase ionsand occurs between the different stages of mass analysis. There are manymethods used to fragment the ions and these can result in differenttypes of fragmentation and thus different information about thestructure and composition of the molecule. The transition ions observedin an LC-SRM-MS spectrum result from several different factors, whichinclude, but are not limited to, the primary sequence, the amount ofinternal energy, the means of introducing the energy, and charge state.Transitions must carry at least one charge to be detected. An ion iscategorized as either a, b or c if the charge is on a transitioncomprising the original N terminus of the peptide, whereas the ion iscategorized as either x, y or z if the charge is on a transitioncomprising the original C terminus of the peptide. A subscript indicatesthe number of residues in the transition (e.g., one peptide residue inx₁, two peptide residues in y₂, and three peptide residues in z₃, etc.).

In a generic peptide repeat unit represented —N—C(O)—C—, an x ion and ana ion resulting from cleavage of the carbonyl-carbon bond (i.e.,C(O)—C). The x ion is an acylium ion, and the a ion is an iminium ion. Ay ion and a b ion result from cleavage of the carbonyl-nitrogen bond(i.e., C(O)—N, also known as the amide bond). In this case, the y ion isan ammonium ion and the b ion is an acylium ion. Finally, a z ion and ac ion result from cleavage of the nitrogen-carbon (i.e., C—N) bond. Thez ion is a carbocation and the c ion is an ammonium ion.

Superscripts are sometimes used to indicate neutral losses in additionto the backbone fragmentation, for example, * for loss of ammonia and °for loss of water. In addition to protons, c ions and y ions mayabstract an additional proton from the precursor peptide. Inelectrospray ionization, tryptic peptides may carry more than onecharge.

Internal transitions arise from double backbone cleavage. These may beformed by a combination of b-type and y-type cleavage (i.e., cleavageproducing b and y ions). Internal cleavage ions may also be formed by acombination of a-type and y-type cleavage. An internal transition with asingle side chain formed by a combination of a-type and y-type cleavageis called an iminium ion (sometimes also referred to as an imonium orimmonium ion). These ions are labeled with the one letter code for thecorresponding amino acid.

Low energy CID (i.e., collision induced dissociation in a triplequadrupole or an ion trap) involves the fragmentation of a peptidecarrying a positive charge, primarily along its backbone, to generateprimarily a, b and y ions.

In one aspect, provided herein is a method for developing peptides andtransitions for a plurality of proteins for a single sample selectedreaction monitoring mass spectrometry (LC-SRM-MS) assay, by: (a)providing a panel of a plurality of proteins; (b) identifying a set ofpeptides for each protein, wherein (i) each peptide in the set ofpeptides corresponds to a transition of said protein; (ii) the peptideshave a monoisotopic mass of 700-5000 Da; and (iii) the peptides do notcontain a cysteine or a methionine; or may contain cysteine ormethionine; (c) identifying a set of transitions for each peptide,wherein (i) the transitions for each peptide have one of the four mostintense b or y transition ions; (ii) the transitions for each peptidehave m/z values of at least 30 m/z above or below those of the precursorion; (iii) the transitions for each peptide do not interfere withtransitions from other peptides; and (iv) the transitions representtransitions due to breakage of peptide bond at different sites of theprotein; and (d) selecting the peptides for each protein that best fitthe criteria of step (b) and the transitions per peptide that best fitthe criteria of step (c); thereby developing peptides and transitionsfor a LC-SRM-MS assay.

By plurality of proteins it is meant at least 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or moreIn certain embodiments, the plurality of proteins can encompass between2 and 10. 10 and 20, 20 and 50, 50 and 100, 100 and 200, or 200 and 500proteins. In other embodiments, the plurality of proteins can encompassbetween 250 and 450; or 300 and 400 proteins.

Trypsin-like proteases cleave peptide bonds following a positivelycharged amino acid (e.g., lysine (K) or arginine (R)). This specificityis driven by the residue which lies at the base of the enzyme's S1pocket (generally a negatively charged aspartic acid or glutamic acid).Accordingly, in one embodiment of the method, the peptides do notinclude any peptide that is bounded by KK, KR, RK or RR, either upstreamof downstream in the corresponding protein sequence. In anotherembodiment, each peptide of said set of peptides is unique to thecorresponding protein.

Post-translational modification (PTM) is the chemical modification of aprotein after its translation. It can include any modification followingtranslation, including cleavage. It is one of the later steps in proteinbiosynthesis, and thus gene expression, for many proteins. It isdesirable to avoid such peptides for the purpose of proteinidentification. Thus, in another embodiment, the peptides do not includepeptides which were observed in post-translational modified forms.

In still another embodiment, each set of peptides is prioritizedaccording to one or more of the following ordered set of criteria: (a)unique peptides first, then non-unique; (b) peptides with no observedpost-translational modifications first, then those observed withpost-translational modifications; (c) peptides within the mass range800-3500 Da first, then those outside of 800-3500 Da; and (d) sorted bydecreasing number of variant residues. In one embodiment, each set ofpeptides is prioritized according to all of the ordered set of criteria.In another embodiment, each prioritized set of peptides contains 1-5peptides.

In certain embodiments, one or more liquid chromatography (LC)purification steps are performed prior to a subsequent LC-SRM-MSanalysis step. Traditional LC analysis relies on the chemicalinteractions between sample components and column packing materials,where laminar flow of the sample through the column is the basis forseparation of the analyte of interest from the test sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess. A variety of column packing materials are available forchromatographic separation of samples, and selection of an appropriateseparation protocol is an empirical process that depends on the samplecharacteristics, the analyte of interest, the interfering substancespresent and their characteristics, etc. Various packing chemistries canbe used depending on the needs (e.g., structure, polarity, andsolubility of compounds being purified). In various embodiments thecolumns are polar, ion exchange (both cation and anion), hydrophobicinteraction, phenyl, C-2, C-8, C-18 columns, polar coating on porouspolymer, or others that are commercially available. Duringchromatography, the separation of materials is effected by variablessuch as choice of eluant (also known as a “mobile phase”), choice ofgradient elution and the gradient conditions, temperature, etc. Incertain embodiments, an analyte may be purified by applying a sample toa column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. As discussed above, suchprocedures may enrich the amount of one or more analytes of interestrelative to one or more other components of the sample.

The following parameters are used to specify an LC-SRM-MS assay of aprotein under a particular LC-SRM-MS system: (1) a tryptic peptide ofthe protein; (2) the retention time (RT) of the peptide; (3) the m/zvalue of the peptide precursor ion; (4) the declustering potential usedto ionize the precursor ion; (5) the m/z value of a fragment iongenerated from the peptide precursor ion; and (6) the collision energy(CE) used to fragment the peptide precursor ion that is optimized forthe particular peptide.

In certain embodiments of the preceding methods, the two best peptidesper protein and the two best transitions per peptide are selected basedon experimental data resulting from LC-SRM-MS analysis of one or more ofthe following experimental samples: a biological disease sample, abiological control sample, and a mixture of synthetic peptides ofinterest. Biological samples include body fluids, tissue samples andcell samples. Body fluid samples can include blood, serum, sputum,genital secretions, cerebrospinal fluid, sweat or excreta such as urine.Body tissue samples can include lung, skin, brain, spine, bone, muscle,epithelial, liver, kidney, pancreas, gastrointestinal tract,cardiovascular tissue, heart or nervous tissue. Biological diseasesamples can include cancer, benign tumors, infected tissue and tissuesubject to trauma. In a particular embodiment, the biological diseaseand biological control samples are processed using an immunodepletionmethod prior to LC-SRM-MS analysis. Immunodepletion involves removal ofone or more proteins through the use of antibodies. Numerousimmunodepletion techniques are known to those of skill in the art. Inanother embodiment, the biological disease and biological controlsamples are processed using an immunocapture method prior to LC-SRM-MSanalysis. Immunocapture involves selection of one or more proteinsthrough the use of antibodies. Numerous immunocapture techniques areknown to those of skill in the art.

To facilitate accurate quantification of the peptide transitions by themethods disclosed herein, a set of isotopically-labeled syntheticversions of the peptides of interest may be added in known amounts tothe sample for use as internal standards. Since the isotopically-labeledpeptides have physical and chemical properties identical to thecorresponding surrogate peptide, they co-elute from the chromatographiccolumn and are easily identifiable on the resultant mass spectrum. Theaddition of the labeled standards may occur before or after proteolyticdigestion. Methods of synthesizing isotopically-labeled peptides will beknown to those of skill in the art. Thus, in another embodiment, theexperimental samples contain internal standard peptides. Otherembodiments may utilize external standards or other expedients forpeptide quantification.

In yet another embodiment, the LC-SRM-MS analysis method specifies amaximum of 7000 transitions, including transitions of the internalstandard peptides and transitions. As used herein, the term “transition”refers to the specific pair of m/z (mass-to-charge) values associatedwith the precursor and transition ions corresponding to a specificpeptide and, therefore, to a specific protein.

In one embodiment of the method, the top two transitions per peptide areselected according to one or more of the following criteria (A): (1) thetransitions exhibit the largest peak areas measured in either of the twobiological experimental samples; (2) the transitions are not interferedwith by other ions; (3) the transitions do not exhibit an elutionprofile that visually differs from those of other transitions of thesame peptide; (4) the transitions are not beyond the detection limit ofboth of the two biological experimental samples; (5) the transitions donot exhibit interferences.

For the mass spectrometric analysis of a particular peptide, thequantities of the peptide transitions in the sample may be determined byintegration of the relevant mass spectral peak areas, as known in theprior art. When isotopically-labeled internal standards are used, asdescribed above, the quantities of the peptide transitions of interestare established via an empirically-derived or predicted relationshipbetween peptide transition quantity (which may be expressed asconcentration) and the area ratio of the peptide transition and internalstandard peaks at specified transitions.

In another embodiment of the method, the top two peptides per proteinare selected according to one or more of the following criteria (B): (1)one or more peptides exhibit two transitions according to criteria (A)and represent the largest combined peak areas of the two transitionsaccording to criteria (A); and (2) one or more peptides exhibit onetransition according to criteria (A) and represent the largest combinedpeak areas of the two transitions according to criteria (A).

Assays

The methods of the present disclosure allow the quantification of highabundance and low abundance plasma proteins that serve as detectablemarkers for various health states (including diseases and disorders),thus forming the basis for assays that can be used to determine thedifferences between normal levels of detectable markers and changes ofsuch detectable markers that are indicative of changes in health status.In one aspect of the invention, provided herein is an assay developedaccording to the foregoing method, and embodiments thereof. In anotheraspect, provided herein is the use of an assay developed according tothe foregoing method, and embodiments thereof, to detect a plurality ofat least 200, 300, or more proteins in a single sample. In a merelyillustration embodiment, 388 proteins in the following table 1 aredetected utilizing the method of present invention.

Of the 388 proteins, the 36 most cooperative proteins are listed inTable 2.

SRM assays for the 388 proteins were developed using standard syntheticpeptide techniques. Of the 388 candidates, SRM assays were successfullydeveloped for 371 candidates. The 371 SRM assays were applied to benignand lung cancer plasma samples to evaluate detection rate in blood. Thesummary of the SRM assay for these 371 proteins is listed in table 3(see also Example III).

Definitions

As used herein, “transition” refers to a pair of m/z values associatedwith a peptide. Normally, labeled synthetic peptides are used as qualitycontrols in SRM assays. However, for very large SRM assays such as the371 protein lung cancer assay, labeled peptides are not feasible.However, correlation techniques (Kearney, Butler et al. 2008) were usedto confirm the identity of protein transitions with high confidence. InFIG. 2 a histogram of the Pearson correlations between every pair oftransitions in the assay is presented. The correlation between a pair oftransitions is obtained from their expression profiles over all samples(143) in the training study detailed below. As expected, transitionsfrom the same peptide are highly correlated. Similarly, transitions fromdifferent peptide fragments of the same protein are also highlycorrelated. In contrast, transitions from different proteins are nothighly correlated. This methodology enables a statistical analysis ofthe quality of a protein's SRM assay. For example, if the correlation oftransitions from two peptides from the same protein is above 0.5 thenthere is less than a 5% probability that the assay is false.

As used herein, a “tryptic peptide” refers to the peptide that is formedby the treatment of a protein with trypsin.

As used herein, “RT” refers to “retention time”, the elapsed timebetween injection and elution of an analyte.

As used herein, “m/z” indicates the mass-to-charge ratio of an ion.

As used herein, “DP” refers to “declustering potential”, a voltagepotential to dissolvate and dissociate ion clusters. It is also known as“fragmentor voltage” or “ion transfer capillary offset voltage”depending on the manufacture.

As used herein, “CE” refers to “collision energy”, the amount of energyprecursor ions receive as they are accelerated into the collision cell.

As used herein, “LC-SRM-MS” is an acronym for “selected reactionmonitoring” and may be used interchangeably with “LC-MRM-MS”.

As used herein, “MS/MS” represents tandem mass spectrometry, which is atype of mass spectrometry involving multiple stages of mass analysiswith some form of fragmentation occurring in between the stages.

As used herein, “ISP” refers to “internal standard peptides”.

As used herein, “HGS” refers to “human gold standard”, which iscomprised of a pool of plasma from healthy individuals.

As used herein, “MGF” refers to “Mascot generic file”. Mascot is asearch engine that uses mass spectrometry data to identify proteins fromprimary sequence databases. A Mascot generic file is a plain text(ASCII) file containing peak list information and, optionally, searchparameters.

Mascot is a web-based tool for deriving protein sequences from massspectrometry data. This data can be acquired from any mass spectrometrytechnique including MALDI-TOF and MS/MS (including LC-SRM-MS) data.Mascot uses a ‘probability-based MOWSE’ algorithm to estimate thesignificance of a match (i.e., that the observed transitions correspondto a particular protein). The total score is the absolute probabilitythat the observed match is a random event. They are reported as−10×LOG10(P), where P is the absolute probability. Lower probabilities,therefore, are reported as higher scores. For example, if the absoluteprobability that an observed match is random is 1×10⁻¹², Mascot reportsit as 120.

The disclosure also provides compositions. These compositions caninclude any of the transition ions described in Table 2. Thesetransition ions exist while peptides derived from the proteins in Table2 are undergoing analysis with LC-SRM-MS. In one embodiment, thecomposition includes any of the transition ions described in Table 2. Inanother embodiment, the composition includes any two transition ionsdescribed in Table 2. In other embodiments, the composition includes,any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300 or 331 transition ions described in Table 2.

In another embodiment, the transition ions correspond with humanproteins including LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR,FRIL, LG3BP, PRDX1, FIBA, and GSLG1. In another embodiment, thetransition ions are derived from human proteins including LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, andGSLG1. These proteins can further include transition ions correspondingwith and/or derived from any number of additional proteins from Table 2.Thus, the composition can include, any additional 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300 or 331 transition ions described in Table 2.

In another embodiment, each of the transition ions in the compositioncorresponds and/or is derived from a different protein. In anotherembodiment, 90% of the transition ions in the composition correspondwith and/or are derived from a protein that no other transition ion inthe composition corresponds. In other embodiments, 80, 70, 60, 50, 40,30, 20, 10 or 0% of the transition ions in the composition correspondand/or are derived from a protein that no other transition ion in thecomposition corresponds.

The compositions described herein included synthetic peptides. Syntheticpeptides can be used as controls for the abundance of proteins they arederived from and/or correspond. In certain embodiments, the abundance ofthe synthetic peptides is defined and the results are compared toLC-SRM-MS results from a peptide found in a sample to the LC-SRM-MSresults in the corresponding synthetic peptide. This allows for thecalculation of the abundance of the peptide in the sample. In certainembodiments, by knowing the abundance of a peptide in a sample, theabundance of the protein it corresponded to is determined.

Synthetic peptides can be generated using any method known in the art.These methods can include recombinant expression techniques such asexpression in bacteria or in vitro expression in eukaryotic cell lysate.These methods can also include solid phase synthesis.

In one embodiment, the composition includes synthetic peptides selectedfrom any of the peptides described in Table 2. In another embodiment,the composition included any two peptides described in Table 2. In otherembodiments, the composition included, any 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or morepeptides described in Table 2.

In another embodiment, the peptides corresponded with human genesincluding LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL,LG3BP, PRDX1, FIBA, and GSLG1 as described in corresponding patentapplication. These genes can further include peptides corresponding withany number of additional genes from Table 2. Thus, the composition caninclude, any additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300 or more peptides describedin Table 2.

In another embodiment, each of the peptides in the composition eachcorresponds with a different protein. In another embodiment, 90% of thepeptides in the composition correspond with a protein that no otherpeptide in the composition corresponds with. In other embodiments, 80,70, 60, 50, 40, 30, 20, 10 or 0% of the peptides in the compositioncorrespond with from a protein that no other peptide in the compositioncorresponds with.

The peptides can be isotopically labeled. The isotopes with which theycan be labeled include ¹³C, ²H, ¹⁵N and ¹⁸O. The peptides can alsoinclude a polar solvent. Polar solvents can include water and mixturesof ethanol and water.

In certain embodiments, the samples described herein are taken frommammals. These mammals include rats, mice, rabbits, dogs, non-humanprimates and humans. Samples can be isolated from any tissue or organ orfrom any bodily fluid. Organs from which samples can be taken includeskin, heart, lung, brain, kidney, liver, pancreas, spleen, testes,ovaries, gall bladder, thymus, thyroid, eye, ear, nose, mouth, tongue,penis, vagina, bladder or larynx. Tissues include nervous tissue,vascular tissue, muscle, bone, gastrointestinal tract, epithelialtissue, fibroblastic tissue, mucous membranes, hair, skin, reproductivetissue and connective tissue. Body fluids and excretions include, blood,serum, saliva, urine, semen, vaginal secretions, excrement, bile, tears,lymph, ear wax, mucous, shed skin, finger nails, toe nails, skin oils,sweat and dandruff.

The relative abundance of one or more of the proteins represented by thetransition ions and synthetic peptides described above can be used todiagnose, determine likelihood of the presence of, develop prognoses forand/or stage various diseases and pathologies. Often the organ, tissueor bodily fluid or excretion from which the sample is taken is distinctfrom the organ, tissue or bodily fluid or excretion involved with thedisease or pathology. For example, the presence of lung cancer can bedetermined from a sample taken from blood. Any type of body fluid may beused in the assays.

Diseases and pathologies that status, diagnosis, presence or prognosiscan be found using the transition ions and/or synthetic peptidesdescribed herein include cancer, metabolic diseases, neurologicaldisorders, infectious diseases and cardiovascular disorders.

EXAMPLES Exemplary Standard Operating Procedure Protein Selection

Proteins known to be over-expressed on the cell surface of lung cancertumors were obtained (through literature searching, experimental data orproprietary databases). This was referred to as set ‘A’.

Proteins known to be over-secreted by lung cancer tumor cells wereobtained (through literature searching, experimental data or proprietarydatabases). This was referred to as set ‘B’. Proteins associated withlung cancer in the literature were mined. This was referred to as set‘C’. Proteins of interest (sets A, B and C are merged resulting in over700 proteins) were assembled. The set of proteins was reduced to a setof 388 proteins (see Table 4) by prioritizing those proteins that havebeen previously detected my LC-MS/MS in blood (serum or plasma).

Selected proteins were then identified by their UniProt protein name andaccession, their Entrez gene symbol and gene name, the isoform accessionand their amino acid sequence. The canonical isoform in UniProt wasselected if a protein has more than one isoform.

Peptide Selection for Synthesis

The five best peptides per protein for LC-SRM-MS assay were selected foras follows. Fully tryptic peptides having a monoisotopic mass of800-3500 mass units, without miscleavages, not containing a cysteine (C)or a methionine (M), without having high miscleavage probability wereselected. Further, any peptide that was bounded by KK, KR, RK or RR(either upstream or downstream) in the corresponding protein sequencewas not selected.

Peptides were selected that were unique to the protein of interest.Peptides were only selected that match only one protein or proteinfamily including analogues of the one protein, when searched in proteindatabases. Further, peptides which were observed in post-translationalmodified forms were not selected. Databases were assessed that showedexpression of the proteins from which the peptides were isolated inhuman blood. Also databases of good quality MS peptides were searched.Peptides that appeared in human blood and were good quality MS peptideswere favored. If these methods did not result in a sufficient number ofpeptides, rules were relaxed in a step wise manner to allow a greaternumber of peptides until a sufficient number was reached. The purity ofthe synthesized peptides was >75% and the amount of material was ≧25 μg.Peptides did not need to be desalted.

The four best transitions per peptide are then selected and optimizedbased on experimental results from a mixture of synthetic peptides.LC-SRM-MS-triggered MS/MS spectra were acquired for each syntheticpeptide, using a QTRAP 5500 instrument. One spectrum for the doubly- andone for the triply-charged precursor ion was collected for each peptideFor the identified peptides (Mascot score ≧15), retention time wasrecorded for the four most intense b or y transition ions. The selectedtransition ions possessed m/z values were at least 30 m/z above or belowthose of the precursor ions; they did not interfere with other syntheticpeptides; and they were transition ions due to breakage of peptide bondat different sites.

If an insufficient percentage of the synthetic peptides were acquired,the steps were repeated. In some cases, the second transition with firstwith theoretical y+ ions with m/z values at least 30 m/z above those ofthe doubly charged precursor ion was selected if an insufficientpercentage was acquired. Peptides that failed to trigger the acquisitionof MS/MS spectrum were discarded.

Collision energy (CE) for each selected transition (See Table 4) wasoptimized.

Exemplary Protein List

The abundance of the following proteins can be assessed substantiallysimultaneously using the MS-LC-SRM-MS system described herein.Transitions from these proteins can be used to diagnose diseasesincluding lung cancer when their abundance is measured in a biologicalspecimen from a subject to be diagnosed for lung cancer. In oneembodiment, the abundances of these proteins are measured in the bloodserum of the subject.

TABLE 1 Sources of Subcellular Evidence for Gene Tissue BiomarkersLocation Presence in UniProt Protein Protein Name Symbol Biomarkers inLiterature (UniProt) Blood 1433B_HUMAN 14-3-3 YWHAB Secreted,LungCancers Cytoplasm. Literature, protein EPI Melanosome. Detectionbeta/alpha Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. 1433E_HUMAN 14-3-3 YWHAE ENDOLungCancers, Cytoplasm (By Literature, protein BenignNodulessimilarity). Detection epsilon Melanosome. Note = Identified by massspectrometry in melanosome fractions from stage I to stage IV.1433S_HUMAN 14-3-3 SFN Secreted, LungCancers Cytoplasm. UniProt, proteinEPI Nucleus (By Literature, sigma similarity). Detection Secreted. Note= May be secreted by a non-classical secretory pathway. 1433T_HUMAN14-3-3 YWHAQ EPI LungCancers, Cytoplasm. Detection protein BenignNodulesNote = In theta neurons, axonally transported to the nerve terminals.1433Z_HUMAN 14-3-3 YWHAZ EPI LungCancers, Cytoplasm. Detection proteinBenignNodules Melanosome. zeta/delta Note = Located to stage I to stageIV melanosomes. 6PGD_HUMAN 6- PGD EPI, ENDO Cytoplasm (By Detectionphosphogluconate similarity). dehydrogenase, decarboxylating A1AG1_HUMANAlpha-1- ORM1 EPI Symptoms Secreted. UniProt, acid Literature,glycoprotein 1 Detection, ABCD1_HUMAN ATP- ABCD1 ENDO PeroxisomeDetection, binding membrane; Prediction cassette Multi-pass sub-familymembrane D member protein. 1 ADA12_HUMAN Disintegrin ADAM12 LungCancers,Isoform 1: Cell UniProt, and BenignNodules, membrane; Detection,metalloproteinase Symptoms Single-pass Prediction domain- type Icontaining membrane protein 12 protein.|Isoform 2: Secreted.|Isoform 3:Secreted (Potential).|Isoform 4: Secreted (Potential). ADML_HUMAN ADMADM LungCancers, Secreted. UniProt, BenignNodules, Literature, SymptomsDetection, Prediction AGR2_HUMAN Anterior AGR2 EPI LungCancers Secreted.UniProt, gradient Endoplasmic Prediction protein 2 reticulum (By homologsimilarity). AIFM1_HUMAN Apoptosis- AIFM1 EPI, ENDO LungCancersMitochondrion Detection, inducing intermembrane Prediction factor 1,space. Nucleus. mitochondrial Note = Translocated to the nucleus uponinduction of apoptosis. ALDOA_HUMAN Fructose- ALDOA Secreted,LungCancers, Literature, bisphosphate EPI Symptoms Detection aldolase AAMPN_HUMAN Aminopeptidase ANPEP EPI, ENDO LungCancers, Cell membrane;UniProt, N BenignNodules, Single-pass Detection Symptoms type IImembrane protein. Cytoplasm, cytosol (Potential). Note = A soluble formhas also been detected. ANGP1_HUMAN Angiopoietin- ANGPT1 LungCancers,Secreted. UniProt, 1 BenignNodules Literature, Prediction ANGP2_HUMANAngiopoietin- ANGPT2 LungCancers, Secreted. UniProt, 2 BenignNodulesLiterature, Prediction APOA1_HUMAN Apolipoprotein APOA1 LungCancers,Secreted. UniProt, A-I BenignNodules, Literature, Symptoms Detection,Prediction APOE_HUMAN Apolipoprotein APOE EPI, ENDO LungCancers,Secreted. UniProt, E BenignNodules, Literature, Symptoms Detection,Prediction ASM3B_HUMAN Acid SMPDL3B EPI, ENDO Secreted (By UniProt,sphingomyelinase- similarity). Prediction like phosphodiesterase 3bAT2A2_HUMAN Sarcoplasmic/ ATP2A2 EPI, ENDO LungCancers, EndoplasmicDetection endoplasmic BenignNodules reticulum reticulum membrane;calcium Multi-pass ATPase 2 membrane protein. Sarcoplasmic reticulummembrane; Multi-pass membrane protein. ATS1_HUMAN A ADAMTS1 LungCancers,Secreted, UniProt, disintegrin BenignNodules, extracellular Literature,and Symptoms space, Prediction metalloproteinase extracellular withmatrix (By thrombospondin similarity). motifs 1 ATS12_HUMAN A ADAMTS12LungCancers Secreted, UniProt, disintegrin extracellular Detection, andspace, Prediction metalloproteinase extracellular with matrix (Bythrombospondin similarity). motifs 12 ATS19_HUMAN A ADAMTS19 LungCancersSecreted, UniProt, disintegrin extracellular Prediction and space,metalloproteinase extracellular with matrix (By thrombospondinsimilarity). motifs 19 BAGE1_HUMAN B BAGE LungCancers Secreted UniProt,melanoma (Potential). Prediction antigen 1 BAGE2_HUMAN B BAGE2LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 2BAGE3_HUMAN B BAGE3 LungCancers Secreted UniProt, melanoma (Potential).Prediction antigen 3 BAGE4_HUMAN B BAGE4 LungCancers Secreted UniProt,melanoma (Potential). Prediction antigen 4 BAGE5_HUMAN B BAGE5LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 5BASP1_HUMAN Brain acid BASP1 Secreted, Cell membrane; Detection solubleEPI Lipid-anchor. protein 1 Cell projection, growth cone. Note =Associated with the membranes of growth cones that form the tips ofelongating axons. BAX_HUMAN Apoptosis BAX EPI LungCancers, IsoformAlpha: UniProt, regulator BenignNodules Mitochondrion Literature, BAXmembrane; Prediction Single-pass membrane protein. Cytoplasm. Note =Colocalizes with 14- 3-3 proteins in the cytoplasm. Under stressconditions, redistributes to the mitochondrion membrane through therelease from JNK- phosphorylated 14-3-3 proteins.|Isoform Beta:Cytoplasm.|Isoform Gamma: Cytoplasm.|Isoform Delta: Cytoplasm(Potential). BDNF_HUMAN Brain- BDNF BenignNodules, Secreted. UniProt,derived Symptoms Literature, neurotrophic Prediction factor BGH3_HUMANTransforming TGFBI LungCancers, Secreted, UniProt, growth BenignNodulesextracellular Detection factor-beta- space, induced extracellularprotein ig- matrix. h3 Note = May be associated both with microfibrilsand with the cell surface. BMP2_HUMAN Bone BMP2 LungCancers, Secreted.UniProt, morphogenetic BenignNodules, Literature protein Symptoms 2BST1_HUMAN ADP- BST1 EPI Symptoms Cell membrane; Detection, ribosylLipid-anchor, Prediction cyclase 2 GPI-anchor. C163A_HUMAN ScavengerCD163 EPI Symptoms Soluble CD163: UniProt, receptor Secreted.|CellDetection cysteine- membrane; rich type 1 Single-pass protein type IM130 membrane protein. Note = Isoform 1 and isoform 2 show a lowersurface expression when expressed in cells. C4BPA_HUMAN C4b- C4BPALungCancers, Secreted. UniProt, binding Symptoms Detection, proteinPrediction alpha chain CAH9_HUMAN Carbonic CA9 LungCancers, Nucleus.UniProt anhydrase BenignNodules, Nucleus, 9 Symptoms nucleolus. Cellmembrane; Single-pass type I membrane protein. Cell projection,microvillus membrane; Single-pass type I membrane protein. Note = Foundon the surface microvilli and in the nucleus, particularly in nucleolus.CALR_HUMAN Calreticulin CALR EPI Symptoms Endoplasmic UniProt, reticulumLiterature, lumen. Detection, Cytoplasm, Prediction cytosol. Secreted,extracellular space, extracellular matrix. Cell surface. Note = Alsofound in cell surface (T cells), cytosol and extracellular matrix.Associated with the lytic granules in the cytolytic T- lymphocytes.CALU_HUMAN Calumenin CALU EPI Symptoms Endoplasmic UniProt, reticulumDetection, lumen. Prediction Secreted. Melanosome. Sarcoplasmicreticulum lumen (By similarity). Note = Identified by mass spectrometryin melanosome fractions from stage I to stage IV. CALX_HUMAN CalnexinCANX Secreted, BenignNodules Endoplasmic UniProt, EPI, ENDO reticulumLiterature, membrane; Detection Single-pass type I membrane protein.Melanosome. Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. CAP7_HUMAN Azurocidin AZU1 EPISymptoms Cytoplasmic Prediction granule. Note = Cytoplasmic granules ofneutrophils. CATB_HUMAN Cathepsin CTSB Secreted LungCancers Lysosome.Literature, B Melanosome. Detection, Note = Identified Prediction bymass spectrometry in melanosome fractions from stage I to stage IV.CATG_HUMAN Cathepsin CTSG Secreted, BenignNodules Cell surface.Detection, G ENDO Prediction CBPB2_HUMAN Carboxypeptidase CPB2LungCancers, Secreted. UniProt, B2 BenignNodules, Detection, SymptomsPrediction CCL22_HUMAN C-C motif CCL22 LungCancers, Secreted. UniProt,chemokine 22 BenignNodules Prediction CD14_HUMAN Monocyte CD14 EPILungCancers, Cell membrane; Literature, differentiation BenignNodules,Lipid-anchor, Detection, antigen Symptoms GPI-anchor. Prediction CD14CD24_HUMAN Signal CD24 LungCancers, Cell membrane; Literature transducerBenignNodules Lipid-anchor, CD24 GPI-anchor. CD2A2_HUMAN Cyclin- CDKN2ALungCancers, Cytoplasm. Literature, dependent BenignNodulesNucleus.|Nucleus, Prediction kinase nucleolus inhibitor 2A, (Bysimilarity). isoform 4 CD38_HUMAN ADP- CD38 EPI, ENDO Symptoms Membrane;UniProt, ribosyl Single-pass Literature cyclase 1 type II membraneprotein. CD40L_HUMAN CD40 CD40LG LungCancers, Cell membrane; UniProt,ligand BenignNodules, Single-pass Literature Symptoms type II membraneprotein.|CD40 ligand, soluble form: Secreted. CD44_HUMAN CD44 CD44 EPILungCancers, Membrane; UniProt, antigen BenignNodules, Single-passLiterature, Symptoms type I Detection, membrane Prediction protein.CD59_HUMAN CD59 CD59 LungCancers, Cell membrane; UniProt, glycoproteinBenignNodules, Lipid-anchor, Literature, Symptoms GPI-anchor. Detection,Secreted. Prediction Note = Soluble form found in a number of tissues.CD97_HUMAN CD97 CD97 EPI, ENDO Symptoms Cell membrane; UniProt antigenMulti-pass membrane protein.|CD97 antigen subunit alpha: Secreted,extracellular space. CDCP1_HUMAN CUB CDCP1 LungCancers Isoform 1: CellUniProt, domain- membrane; Prediction containing Single-pass protein 1membrane protein (Potential). Note = Shedding may also lead to a solublepeptide.|Isoform 3: Secreted. CDK4_HUMAN Cell CDK4 LungCancers,Literature division Symptoms protein kinase 4 CEAM5_HUMANCarcinoembryonic CEACAM5 EPI LungCancers, Cell membrane; Literature,antigen- BenignNodules, Lipid-anchor, Prediction related cell SymptomsGPI-anchor. adhesion molecule 5 CEAM8_HUMAN Carcinoembryonic CEACAM8 EPILungCancers Cell membrane; Detection, antigen- Lipid-anchor, Predictionrelated cell GPI-anchor. adhesion molecule 8 CERU_HUMAN Ceruloplasmin CPEPI LungCancers, Secreted. UniProt, Symptoms Literature, Detection,Prediction CH10_HUMAN 10 kDa HSPE1 ENDO LungCancers MitochondrionLiterature, heat shock matrix. Detection, protein, Predictionmitochondrial CH60_HUMAN 60 kDa HSPD1 Secreted, LungCancers,Mitochondrion Literature, heat shock EPI, ENDO Symptoms matrix.Detection protein, mitochondrial CKAP4_HUMAN Cytoskeleton- CKAP4 EPI,ENDO LungCancers Endoplasmic UniProt associated reticulum-Golgi protein4 intermediate compartment membrane; Single-pass membrane protein(Potential). CL041_HUMAN Uncharacterized C12orf41 ENDO Predictionprotein C12orf41 CLCA1_HUMAN Calcium- CLCA1 LungCancers, Secreted,UniProt, activated BenignNodules extracellular Prediction chloridespace. Cell channel membrane; regulator 1 Peripheral membrane protein;Extracellular side. Note = Protein that remains attached to the plasmamembrane appeared to be predominantly localized to microvilli.CLIC1_HUMAN Chloride CLIC1 EPI Nucleus. UniProt, intracellular NucleusLiterature, channel membrane; Detection protein 1 Single-pass membraneprotein (Probable). Cytoplasm. Cell membrane; Single-pass membraneprotein (Probable). Note = Mostly in the nucleus including in thenuclear membrane. Small amount in the cytoplasm and the plasma membrane.Exists both as soluble cytoplasmic protein and as membrane protein withprobably a single transmembrane domain. CLUS_HUMAN Clusterin CLU EPI,ENDO LungCancers, Secreted. UniProt, BenignNodules, Literature, SymptomsDetection, Prediction CMGA_HUMAN Chromogranin- CHGA LungCancers,Secreted. UniProt, A BenignNodules Note = Literature, Neuroendocrine andDetection, endocrine Prediction secretory granules. CNTN1_HUMANContactin-1 CNTN1 LungCancers Isoform 1: Cell Detection, membrane;Prediction Lipid-anchor, GPI- anchor; Extracellular side.|Isoform 2:Cell membrane; Lipid-anchor, GPI- anchor; Extracellular side.CO4A1_HUMAN Collagen COL4A1 LungCancers Secreted, UniProt, alpha-1(IV)extracellular Detection, chain space, Prediction extracellular matrix,basement membrane. CO5A2_HUMAN Collagen COL5A2 LungCancers Secreted,UniProt, alpha-2(V) extracellular Detection, chain space, Predictionextracellular matrix (By similarity). CO6A3_HUMAN Collagen COL6A3Secreted Symptoms Secreted, UniProt, alpha-3(VI) extracellularDetection, chain space, Prediction extracellular matrix (By similarity).COCA1_HUMAN Collagen COL12A1 ENDO LungCancers, Secreted, UniProt, alpha-Symptoms extracellular Prediction 1(XII) space, chain extracellularmatrix (By similarity). COF1_HUMAN Cofilin-1 CFL1 Secreted, LungCancers,Nucleus matrix. Detection, EPI BenignNodules Cytoplasm, Predictioncytoskeleton. Note = Almost completely in nucleus in cells exposed toheat shock or 10% dimethyl sulfoxide. COIA1_HUMAN Collagen COL18A1LungCancers, Secreted, UniProt, alpha- BenignNodules extracellularLiterature, 1(XVIII) space, Detection, chain extracellular Predictionmatrix (By similarity). COX5A_HUMAN Cytochrome COX5A Secreted,Mitochondrion Prediction c oxidase ENDO inner subunit 5A, membrane.mitochondrial CRP_HUMAN C-reactive CRP LungCancers, Secreted. UniProt,protein BenignNodules, Literature, Symptoms Detection, PredictionCS051_HUMAN UPF0470 C19orf51 ENDO Prediction protein C19orf51 CSF1_HUMANMacrophage CSF1 LungCancers, Cell membrane; UniProt, colony-BenignNodules Single-pass Literature, stimulating membrane Detectionfactor 1 protein (By similarity).|Processed macrophage colony-stimulating factor 1: Secreted, extracellular space (By similarity).CSF2_HUMAN Granulocyte- CSF2 LungCancers, Secreted. UniProt, macrophageBenignNodules Literature, colony- Prediction stimulating factorCT085_HUMAN Uncharacterized C20orf85 LungCancers, Prediction proteinBenignNodules C20orf85 CTGF_HUMAN Connective CTGF LungCancers, Secreted,UniProt, tissue BenignNodules extracellular Literature, growth space,Detection, factor extracellular Prediction matrix (By similarity).Secreted (By similarity). CYR61_HUMAN Protein CYR61 LungCancers,Secreted. UniProt, CYR61 BenignNodules Prediction CYTA_HUMAN Cystatin-ACSTA LungCancers Cytoplasm. Literature, Detection CYTB_HUMAN Cystatin-BCSTB Secreted Cytoplasm. Literature, Nucleus. Detection DDX17_HUMANProbable DDX17 ENDO LungCancers, Nucleus. Detection, ATP- BenignNodulesPrediction dependent RNA helicase DDX17 DEFB1_HUMAN Beta- DEFB1LungCancers, Secreted. UniProt, defensin 1 BenignNodules PredictionDESP_HUMAN Desmoplakin DSP EPI, ENDO LungCancers Cell junction,Detection desmosome. Cytoplasm, cytoskeleton. Note = Innermost portionof the desmosomal plaque. DFB4A_HUMAN Beta- DEFB4A LungCancers,Secreted. UniProt defensin BenignNodules 4A DHI1L_HUMAN HydroxysteroidHSD11B1L LungCancers Secreted UniProt, 11- (Potential). Prediction beta-dehydrogenase 1-like protein DMBT1_HUMAN Deleted in DMBT1 LungCancers,Secreted (By UniProt, malignant BenignNodules similarity). Detection,brain Note = Some Prediction tumors 1 isoforms may protein be membrane-bound. Localized to the lumenal aspect of crypt cells in the smallintestine. In the colon, seen in the lumenal aspect of surfaceepithelial cells. Formed in the ducts of von Ebner gland, and releasedinto the fluid bathing the taste buds contained in the taste papillae(By similarity). DMKN_HUMAN Dermokine DMKN LungCancers Secreted.UniProt, Detection, Prediction DPP4_HUMAN Dipeptidyl DPP4 EPILungCancers, Dipeptidyl UniProt, peptidase 4 BenignNodules, peptidase 4Detection Symptoms soluble form: Secreted.|Cell membrane; Single-passtype II membrane protein. DSG2_HUMAN Desmoglein- DSG2 ENDO Symptoms Cellmembrane; UniProt, 2 Single-pass Detection type I membrane protein. Celljunction, desmosome. DX39A_HUMAN ATP- DDX39A EPI Nucleus (By Predictiondependent similarity). RNA helicase DDX39A DX39B_HUMAN SpliceosomeDDX39B EPI Nucleus. Prediction RNA Nucleus helicase speckle. DDX39BDYRK2_HUMAN Dual DYRK2 ENDO LungCancers Cytoplasm. Literaturespecificity Nucleus. tyrosine- Note = Translocates phosphorylation- intothe regulated nucleus kinase 2 following DNA damage. EDN2_HUMANEndothelin-2 EDN2 LungCancers Secreted. UniProt, Prediction EF1A1_HUMANElongation EEF1A1 Secreted, LungCancers, Cytoplasm. Detection factor 1-EPI BenignNodules alpha 1 EF1D_HUMAN Elongation EEF1D Secreted,LungCancers Prediction factor 1- EPI delta EF2_HUMAN Elongation EEF2Secreted, Cytoplasm. Literature, factor 2 EPI Detection EGF_HUMAN Pro-EGF LungCancers, Membrane; UniProt, epidermal BenignNodules, Single-passLiterature growth Symptoms type I factor membrane protein. EGFL6_HUMANEpidermal EGFL6 LungCancers Secreted, UniProt, growth extracellularDetection, factor-like space, Prediction protein 6 extracellular matrix,basement membrane (By similarity). ENOA_HUMAN Alpha- ENO1 Secreted,LungCancers, Cytoplasm. Cell Literature, enolase EPI, ENDOBenignNodules, membrane. Detection, Symptoms Cytoplasm, Predictionmyofibril, sarcomere, M- band. Note = Can translocate to the plasmamembrane in either the homodimeric (alpha/alpha) or heterodimeric(alpha/gamma) form. ENO1 is localized to the M- band.|Isoform MBP-1:Nucleus. ENOG_HUMAN Gamma- ENO2 EPI LungCancers, Cytoplasm (ByLiterature, enolase Symptoms similarity). Cell Detection, membrane (ByPrediction similarity). Note = Can translocate to the plasma membrane ineither the homodimeric (alpha/alpha) or heterodimeric (alpha/gamma) form(By similarity). ENOX2_HUMAN Ecto-NOX ENOX2 LungCancers Cell membrane.UniProt, disulfide- Secreted, Detection thiol extracellular exchangerspace. 2 Note = Extracellular and plasma membrane- associated.ENPL_HUMAN Endoplasmin HSP90B1 Secreted, LungCancers, EndoplasmicLiterature, EPI, ENDO BenignNodules, reticulum Detection, Symptomslumen. Prediction Melanosome. Note = Identified by mass spectrometry inmelanosome fractions from stage I to stage IV. EPHB6_HUMAN Ephrin EPHB6LungCancers Membrane; UniProt, type-B Single-pass Literature receptor 6type I membrane protein.|Isoform 3: Secreted (Probable). EPOR_HUMANErythropoietin EPOR LungCancers, Cell membrane; UniProt, receptorBenignNodules, Single-pass Literature, Symptoms type I Detectionmembrane protein.|Isoform EPOR-S: Secreted. Note = Secreted and locatedto the cell surface. ERBB3_HUMAN Receptor ERBB3 LungCancers, Isoform 1:Cell UniProt, tyrosine- BenignNodules membrane; Literature, proteinSingle-pass Prediction kinase type I erbB-3 membrane protein.|Isoform 2:Secreted. EREG_HUMAN Proepiregulin EREG LungCancers Epiregulin: UniProtSecreted, extracellular space.|Proepiregulin: Cell membrane; Single-passtype I membrane protein. ERO1A_HUMAN ERO1-like ERO1L Secreted, SymptomsEndoplasmic Prediction protein EPI, ENDO reticulum alpha membrane;Peripheral membrane protein; Lumenal side. Note = The association withERP44 is essential for its retention in the endoplasmic reticulum.ESM1_HUMAN Endothelial ESM1 LungCancers, Secreted. UniProt, cell-BenignNodules Prediction specific molecule 1 EZRI_HUMAN Ezrin EZRSecreted LungCancers, Apical cell Literature, BenignNodules membrane;Detection, Peripheral Prediction membrane protein; Cytoplasmic side.Cell projection. Cell projection, microvillus membrane; Peripheralmembrane protein; Cytoplasmic side. Cell projection, ruffle membrane;Peripheral membrane protein; Cytoplasmic side. Cytoplasm, cell cortex.Cytoplasm, cytoskeleton. Note = Localization to the apical membrane ofparietal cells depends on the interaction with MPP5. Localizes to cellextensions and peripheral processes of astrocytes (By similarity).Microvillar peripheral membrane protein (cytoplasmic side). F10A1_HUMANHsc70- ST13 EPI Cytoplasm (By Detection, interactingsimilarity).|Cytoplasm Prediction protein (Probable). FAM3C_HUMANProtein FAM3C EPI, ENDO Secreted UniProt, FAM3C (Potential). DetectionFAS_HUMAN Fatty acid FASN EPI LungCancers, Cytoplasm. Literature,synthase BenignNodules, Melanosome. Detection Symptoms Note = Identifiedby mass spectrometry in melanosome fractions from stage I to stage IV.FCGR1_HUMAN High FCGR1A EPI LungCancers, Cell membrane; UniProt affinityBenignNodules, Single-pass immunoglobulin Symptoms type I gamma Fcmembrane receptor I protein. Note = Stabilized at the cell membranethrough interaction with FCER1G. FGF10_HUMAN Fibroblast FGF10LungCancers Secreted UniProt, growth (Potential). Prediction factor 10FGF2_HUMAN Heparin- FGF2 , LungCancers, Literature bindingBenignNodules, growth Symptoms factor 2 FGF7_HUMAN Keratinocyte FGF7LungCancers, Secreted. UniProt, growth BenignNodules Literature, factorPrediction FGF9_HUMAN Glia- FGF9 LungCancers Secreted. UniProt,activating Literature, factor Prediction FGFR2_HUMAN Fibroblast FGFR2LungCancers, Cell membrane; UniProt, growth BenignNodules Single-passLiterature, factor type I Prediction receptor 2 membraneprotein.|Isoform 14: Secreted.|Isoform 19: Secreted. FGFR3_HUMANFibroblast FGFR3 LungCancers Membrane; UniProt, growth Single-passLiterature, factor type I Prediction receptor 3 membrane protein.FGL2_HUMAN Fibroleukin FGL2 BenignNodules, Secreted. UniProt, SymptomsDetection, Prediction FHIT_HUMAN Bis(5′- FHIT LungCancers, Cytoplasm.Literature adenosyl)- BenignNodules, triphosphatase Symptoms FIBA_HUMANFibrinogen FGA LungCancers, Secreted. UniProt, alpha chainBenignNodules, Literature, Symptoms Detection, Prediction FINC_HUMANFibronectin FN1 Secreted, LungCancers, Secreted, UniProt, EPI, ENDOBenignNodules, extracellular Literature, Symptoms space, Detection,extracellular Prediction matrix. FKB11_HUMAN Peptidyl- FKBP11 EPI, ENDOMembrane; UniProt, prolyl cis- Single-pass Prediction trans membraneisomerase protein FKBP11 (Potential). FOLH1_HUMAN Glutamate FOLH1 ENDOLungCancers, Cell membrane; UniProt, carboxypeptidase SymptomsSingle-pass Literature 2 type II membrane protein.|Isoform PSMA′:Cytoplasm. FOLR1_HUMAN Folate FOLR1 LungCancers Cell membrane; UniProtreceptor Lipid-anchor, alpha GPI-anchor. Secreted (Probable).FOXA2_HUMAN Hepatocyte FOXA2 LungCancers Nucleus. Detection, nuclearPrediction factor 3- beta FP100_HUMAN Fanconi C17orf70 ENDO SymptomsNucleus. Prediction anemia- associated protein of 100 kDa FRIH_HUMANFerritin FTH1 EPI LungCancers, Literature, heavy BenignNodulesDetection, chain Prediction FRIL_HUMAN Ferritin FTL Secreted,BenignNodules, Literature, light chain EPI, ENDO Symptoms DetectionG3P_HUMAN Glyceraldehyde- GAPDH Secreted, LungCancers, Cytoplasm.Detection 3- EPI, ENDO BenignNodules, Cytoplasm, phosphate Symptomsperinuclear dehydrogenase region. Membrane. Note = Postnuclear andPerinuclear regions. G6PD_HUMAN Glucose-6- G6PD Secreted, LungCancers,Literature, phosphate EPI Symptoms Detection 1- dehydrogenase G6PI_HUMANGlucose-6- GPI Secreted, Symptoms Cytoplasm. UniProt, phosphate EPISecreted. Literature, isomerase Detection GA2L1_HUMAN GAS2-like GAS2L1ENDO Cytoplasm, Prediction protein 1 cytoskeleton (Probable).GALT2_HUMAN Polypeptide GALNT2 EPI, ENDO Golgi UniProt, N- apparatus,Detection acetylgalactosaminyl- Golgi stack transferase 2 membrane;Single-pass type II membrane protein. Secreted. Note = Residespreferentially in the trans and medial parts of the Golgi stack. Asecreted form also exists. GAS6_HUMAN Growth GAS6 LungCancers Secreted.UniProt, arrest- Detection, specific Prediction protein 6 GDIR2_HUMANRho GDP- ARHGDIB EPI Cytoplasm. Detection dissociation inhibitor 2GELS_HUMAN Gelsolin GSN LungCancers, Isoform 2: UniProt, BenignNodulesCytoplasm, Literature, cytoskeleton.|Isoform Detection, 1: PredictionSecreted. GGH_HUMAN Gamma- GGH LungCancers Secreted, UniProt, glutamylextracellular Detection, hydrolase space. Prediction Lysosome.Melanosome. Note = While its intracellular location is primarily thelysosome, most of the enzyme activity is secreted. Identified by massspectrometry in melanosome fractions from stage I to stage IV.GPC3_HUMAN Glypican-3 GPC3 LungCancers, Cell membrane; UniProt, SymptomsLipid-anchor, Literature, GPI-anchor; Prediction Extracellular side (Bysimilarity).|Secreted glypican-3: Secreted, extracellular space (Bysimilarity). GRAN_HUMAN Grancalcin GCA EPI Cytoplasm. PredictionCytoplasmic granule membrane; Peripheral membrane protein; Cytoplasmicside. Note = Primarily cytosolic in the absence of calcium or magnesiumions. Relocates to granules and other membranes in response to elevatedcalcium and magnesium levels. GREB1_HUMAN Protein GREB1 ENDO Membrane;UniProt, GREB1 Single-pass Prediction membrane protein (Potential).GREM1_HUMAN Gremlin-1 GREM1 LungCancers, Secreted UniProt, BenignNodules(Probable). Prediction GRP_HUMAN Gastrin- GRP LungCancers, Secreted.UniProt, releasing Symptoms Prediction peptide GRP78_HUMAN 78 kDa HSPA5Secreted, LungCancers, Endoplasmic Detection, glucose- EPI, ENDOBenignNodules reticulum Prediction regulated lumen. protein Melanosome.Note = Identified by mass spectrometry in melanosome fractions fromstage I to stage IV. GSLG1_HUMAN Golgi GLG1 EPI, ENDO BenignNodulesGolgi apparatus UniProt apparatus membrane; protein 1 Single-pass type Imembrane protein. GSTP1_HUMAN Glutathione GSTP1 Secreted LungCancers,Literature, S- BenignNodules, Detection, transferase Symptoms PredictionP GTR1_HUMAN Solute SLC2A1 EPI, ENDO LungCancers, Cell membrane;Literature carrier BenignNodules, Multi-pass family 2, Symptoms membranefacilitated protein (By glucose similarity). transporter Melanosome.member 1 Note = Localizes primarily at the cell surface (By similarity).Identified by mass spectrometry in melanosome fractions from stage I tostage IV. GTR3_HUMAN Solute SLC2A3 EPI Membrane; Detection carrierMulti-pass family 2, membrane facilitated protein. glucose transportermember 3 H2A1_HUMAN Histone HIST1H2AG Secreted Nucleus. Detection, H2Atype 1 Prediction H2A1B_HUMAN Histone HIST1H2AB Secreted Nucleus.Detection, H2A type Prediction 1-B/E H2A1C_HUMAN Histone HIST1H2ACSecreted Nucleus. Literature, H2A type Detection, 1-C PredictionH2A1D_HUMAN Histone HIST1H2AD Secreted Nucleus. Detection, H2A typePrediction 1-D HG2A_HUMAN HLA class CD74 LungCancers, Membrane; UniProt,II BenignNodules, Single-pass Literature histocompatibility Symptomstype II antigen membrane gamma chain protein (Potential). HGF_HUMANHepatocyte HGF LungCancers, Literature, growth BenignNodules, Predictionfactor Symptoms HMGA1_HUMAN High HMGA1 LungCancers, Nucleus. Literaturemobility BenignNodules, group Symptoms protein HMG- I/HMG-Y HPRT_HUMANHypoxanthine- HPRT1 EPI Cytoplasm. Detection, guanine Predictionphosphoribosyl- transferase HPSE_HUMAN Heparanase HPSE LungCancers,Lysosome UniProt, BenignNodules, membrane; Prediction SymptomsPeripheral membrane protein. Secreted. Note = Secreted, internalised andtransferred to late endosomes/lysosomes as a proheparanase. Inlysosomes, it is processed into the active form, the heparanase. Theuptake or internalisation of proheparanase is mediated by HSPGs. Heparinappears to be a competitor and retain proheparanase in the extracellularmedium. HPT_HUMAN Haptoglobin HP LungCancers, Secreted. UniProt,BenignNodules, Literature, Symptoms Detection, Prediction HS90A_HUMANHeat shock HSP90AA1 Secreted, LungCancers, Cytoplasm. Literature,protein EPI Symptoms Melanosome. Detection HSP 90- Note = Identifiedalpha by mass spectrometry in melanosome fractions from stage I to stageIV. HS90B_HUMAN Heat shock HSP90AB1 Secreted, LungCancers Cytoplasm.Literature, protein EPI Melanosome. Detection HSP 90- Note = Identifiedbeta by mass spectrometry in melanosome fractions from stage I to stageIV. HSPB1_HUMAN Heat shock HSPB1 Secreted, LungCancers, Cytoplasm.Literature, protein EPI BenignNodules Nucleus. Detection, beta-1Cytoplasm, Prediction cytoskeleton, spindle. Note = Cytoplasmic ininterphase cells. Colocalizes with mitotic spindles in mitotic cells.Translocates to the nucleus during heat shock. HTRA1_HUMAN Serine HTRA1LungCancers Secreted. UniProt, protease Prediction HTRA1 HXK1_HUMANHexokinase- HK1 ENDO Symptoms Mitochondrion Literature, 1 outerDetection membrane. Note = Its hydrophobic N- terminal sequence may beinvolved in membrane binding. HYAL2_HUMAN Hyaluronidase- HYAL2LungCancers Cell membrane; Prediction 2 Lipid-anchor, GPI-anchor.HYOU1_HUMAN Hypoxia HYOU1 EPI, ENDO Symptoms Endoplasmic Detection up-reticulum regulated lumen. protein 1 IBP2_HUMAN Insulin-like IGFBP2LungCancers Secreted. UniProt, growth Literature, factor- Detection,binding Prediction protein 2 IBP3_HUMAN Insulin-like IGFBP3 LungCancers,Secreted. UniProt, growth BenignNodules, Literature, factor- SymptomsDetection, binding Prediction protein 3 ICAM1_HUMAN Intercellular ICAM1LungCancers, Membrane; UniProt, adhesion BenignNodules, Single-passLiterature, molecule 1 Symptoms type I Detection membrane protein.ICAM3_HUMAN Intercellular ICAM3 EPI, ENDO LungCancers, Membrane;UniProt, adhesion BenignNodules, Single-pass Detection molecule 3Symptoms type I membrane protein. IDHP_HUMAN Isocitrate IDH2 Secreted,Mitochondrion. Prediction dehydrogenase ENDO [NADP], mitochondrialIF4A1_HUMAN Eukaryotic EIF4A1 Secreted, Detection, initiation EPI, ENDOPrediction factor 4A-I IGF1_HUMAN Insulin-like IGF1 LungCancers,Secreted.|Secreted. UniProt, growth BenignNodules, Literature, factor ISymptoms Detection, Prediction IKIP_HUMAN Inhibitor of IKIP ENDOSymptoms Endoplasmic UniProt, nuclear reticulum Prediction factormembrane; kappa-B Single-pass kinase- membrane interacting protein.protein Note = Isoform 4 deletion of the hydrophobic, or transmembraneregion between AA 45-63 results in uniform distribution troughout thecell, suggesting that this region is responsible for endoplasmicreticulum localization. IL18_HUMAN Interleukin- IL18 LungCancers,Secreted. UniProt, 18 BenignNodules, Literature, Symptoms PredictionIL19_HUMAN Interleukin- IL19 LungCancers Secreted. UniProt, 19Detection, Prediction IL22_HUMAN Interleukin- IL22 LungCancers,Secreted. UniProt, 22 BenignNodules Prediction IL32_HUMAN Interleukin-IL32 LungCancers, Secreted. UniProt, 32 BenignNodules PredictionIL7_HUMAN Interleukin- IL7 LungCancers, Secreted. UniProt, 7BenignNodules Literature, Prediction IL8_HUMAN Interleukin- IL8LungCancers, Secreted. UniProt, 8 BenignNodules, Literature SymptomsILEU_HUMAN Leukocyte SERPINB1 Secreted, Cytoplasm (By Detection,elastase EPI similarity). Prediction inhibitor ILK_HUMAN Integrin- ILKSecreted LungCancers, Cell junction, Literature, linked BenignNodules,focal adhesion. Detection protein Symptoms Cell membrane; kinasePeripheral membrane protein; Cytoplasmic side. INHBA_HUMAN Inhibin INHBALungCancers, Secreted. UniProt, beta A BenignNodules Literature, chainPrediction ISLR_HUMAN Immunoglobulin ISLR LungCancers Secreted UniProt,superfamily (Potential). Detection, containing Prediction leucine- richrepeat protein ITA5_HUMAN Integrin ITGA5 EPI LungCancers, Membrane;UniProt, alpha-5 BenignNodules, Single-pass Literature, Symptoms type IDetection membrane protein. ITAM_HUMAN Integrin ITGAM EPI, ENDOLungCancers, Membrane; UniProt, alpha-M BenignNodules, Single-passLiterature Symptoms type I membrane protein. K0090_HUMAN UncharacterizedKIAA0090 EPI Symptoms Membrane; UniProt, protein Single-pass PredictionKIAA0090 type I membrane protein (Potential). K1C18_HUMAN Keratin, KRT18Secreted LungCancers, Cytoplasm, Literature, type I BenignNodulesperinuclear Detection, cytoskeletal region. Prediction 18 K1C19_HUMANKeratin, KRT19 LungCancers, Literature, type I BenignNodules Detection,cytoskeletal Prediction 19 K2C8_HUMAN Keratin, KRT8 EPI LungCancersCytoplasm. Literature, type II Detection cytoskeletal 8 KIT_HUMANMast/stem KIT LungCancers Membrane; UniProt, cell growth Single-passLiterature, factor type I Detection receptor membrane protein.KITH_HUMAN Thymidine TK1 LungCancers Cytoplasm. Literature, kinase,Prediction cytosolic KLK11_HUMAN Kallikrein- KLK11 LungCancers Secreted.UniProt, 11 Literature, Prediction KLK13_HUMAN Kallikrein- KLK13LungCancers Secreted UniProt, 13 (Probable). Literature, Detection,Prediction KLK14_HUMAN Kallikrein- KLK14 LungCancers, Secreted, UniProt,14 Symptoms extracellular Literature, space. Prediction KLK6_HUMANKallikrein- KLK6 LungCancers, Secreted. UniProt, 6 BenignNodules,Nucleus, Literature, Symptoms nucleolus. Detection, Cytoplasm.Prediction Mitochondrion. Microsome. Note = In brain, detected in thenucleus of glial cells and in the nucleus and cytoplasm of neurons.Detected in the mitochondrial and microsomal fractions of HEK-293 cellsand released into the cytoplasm following cell stress. KNG1_HUMANKininogen- KNG1 LungCancers, Secreted, UniProt, 1 BenignNodules,extracellular Detection, Symptoms space. Prediction KPYM_HUMAN PyruvatePKM2 Secreted, LungCancers, Cytoplasm. Literature, kinase EPI SymptomsNucleus. Detection isozymes Note = Translocates M1/M2 to the nucleus inresponse to different apoptotic stimuli. Nuclear translocation issufficient to induce cell death that is caspase independent,isoform-specific and independent of its enzymatic actvity. KRT35_HUMANKeratin, KRT35 ENDO Detection, type I Prediction cuticular Ha5LAMB2_HUMAN Laminin LAMB2 ENDO LungCancers, Secreted, UniProt, subunitSymptoms extracellular Detection, beta-2 space, Prediction extracellularmatrix, basement membrane. Note = S-laminin is concentrated in thesynaptic cleft of the neuromuscular junction. LDHA_HUMAN L-lactate LDHASecreted, LungCancers Cytoplasm. Literature, dehydrogenase EPI, ENDODetection, A chain Prediction LDHB_HUMAN L-lactate LDHB EPI LungCancersCytoplasm. Detection, dehydrogenase Prediction B chain LEG1_HUMANGalectin-1 LGALS1 Secreted LungCancers Secreted, UniProt, extracellularDetection space, extracellular matrix. LEG3_HUMAN Galectin-3 LGALS3LungCancers, Nucleus. Literature, BenignNodules Note = CytoplasmicDetection, in Prediction adenomas and carcinomas. May be secreted by anon- classical secretory pathway and associate with the cell surface.LEG9_HUMAN Galectin-9 LGALS9 ENDO Symptoms Cytoplasm (By UniProtsimilarity). Secreted (By similarity). Note = May also be secreted by anon-classical secretory pathway (By similarity). LG3BP_HUMAN Galectin-3-LGALS3BP Secreted LungCancers, Secreted. UniProt, binding BenignNodules,Secreted, Literature, protein Symptoms extracellular Detection, space,Prediction extracellular matrix. LPLC3_HUMAN Long C20orf185 LungCancersSecreted (By UniProt, palate, lung similarity). Prediction and nasalCytoplasm. epithelium Note = According carcinoma- to associated PubMed:protein 3 12837268 it is cytoplasmic. LPLC4_HUMAN Long C20orf186LungCancers Secreted (By UniProt, palate, lung similarity). Predictionand nasal Cytoplasm. epithelium carcinoma- associated protein 4LPPRC_HUMAN Leucine- LRPPRC Secreted, LungCancers, Mitochondrion.Prediction rich PPR ENDO Symptoms Nucleus, motif- nucleoplasm.containing Nucleus inner protein, membrane. mitochondrial Nucleus outermembrane. Note = Seems to be predominantly mitochondrial. LRP1_HUMANProlow- LRP1 EPI LungCancers, Low-density UniProt, density Symptomslipoprotein Detection lipoprotein receptor-related receptor- protein 185 related kDa subunit: protein 1 Cell membrane; Single-pass type Imembrane protein. Membrane, coated pit.|Low- density lipoproteinreceptor-related protein 1 515 kDa subunit: Cell membrane; Peripheralmembrane protein; Extracellular side. Membrane, coated pit.|Low- densitylipoprotein receptor-related protein 1 intracellular domain: Cytoplasm.Nucleus. Note = After cleavage, the intracellular domain (LRPICD) isdetected both in the cytoplasm and in the nucleus. LUM_HUMAN Lumican LUMSecreted, LungCancers, Secreted, UniProt, EPI BenignNodules,extracellular Detection, Symptoms space, Prediction extracellular matrix(By similarity). LY6K_HUMAN Lymphocyte LY6K LungCancers, Secreted.UniProt, antigen Symptoms Cytoplasm. Cell Prediction 6K membrane; Lipid-anchor, GPI-anchor (Potential). LYAM2_HUMAN E-selectin SELE LungCancers,Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms typeI Detection membrane protein. LYAM3_HUMAN P-selectin SELP LungCancers,Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms typeI Detection membrane protein. LYOX_HUMAN Protein- LOX LungCancers,Secreted, UniProt, lysine 6- BenignNodules extracellular Detection,oxidase space. Prediction LYPD3_HUMAN Ly6/PLAUR LYPD3 LungCancers Cellmembrane; Detection, domain- Lipid-anchor, Prediction containingGPI-anchor. protein 3 MAGA4_HUMAN Melanoma- MAGEA4 LungCancersLiterature, associated Prediction antigen 4 MASP1_HUMAN Mannan- MASP1LungCancers, Secreted. UniProt, binding Symptoms Detection, lectinPrediction serine protease 1 MDHC_HUMAN Malate MDH1 Secreted Cytoplasm.Literature, dehydrogenase, Detection, cytoplasmic Prediction MDHM_HUMANMalate MDH2 ENDO LungCancers Mitochondrion Detection, dehydrogenase,matrix. Prediction mitochondrial MIF_HUMAN Macrophage MIF SecretedLungCancers, Secreted. UniProt, migration BenignNodules, Cytoplasm.Literature, inhibitory Symptoms Note = Does not Prediction factor have acleavable signal sequence and is secreted via a specialized, non-classical pathway. Secreted by macrophages upon stimulation by bacteriallipopolysaccharide (LPS), or by M. tuberculosis antigens. MLH1_HUMAN DNAMLH1 ENDO LungCancers, Nucleus. Literature mismatch BenignNodules,repair Symptoms protein Mlh1 MMP1_HUMAN Interstitial MMP1 LungCancers,Secreted, UniProt, collagenase BenignNodules, extracellular Literature,Symptoms space, Prediction extracellular matrix (Probable). MMP11_HUMANStromelysin- MMP11 LungCancers, Secreted, UniProt, 3 Symptomsextracellular Literature, space, Prediction extracellular matrix(Probable). MMP12_HUMAN Macrophage MMP12 LungCancers, Secreted, UniProt,metalloelastase BenignNodules, extracellular Literature, Symptoms space,Prediction extracellular matrix (Probable). MMP14_HUMAN Matrix MMP14ENDO LungCancers, Membrane; UniProt, metalloproteinase- BenignNodules,Single-pass Literature, 14 Symptoms type I Detection membrane protein(Potential). Melanosome. Note = Identified by mass spectrometry inmelanosome fractions from stage I to stage IV. MMP2_HUMAN 72 kDa MMP2LungCancers, Secreted, UniProt, type IV BenignNodules, extracellularLiterature, collagenase Symptoms space, Detection, extracellularPrediction matrix (Probable). MMP26_HUMAN Matrix MMP26 LungCancersSecreted, UniProt, metalloproteinase- extracellular Prediction 26 space,extracellular matrix. MMP7_HUMAN Matrilysin MMP7 LungCancers, Secreted,UniProt, BenignNodules, extracellular Literature, Symptoms space,Prediction extracellular matrix (Probable). MMP9_HUMAN Matrix MMP9LungCancers, Secreted, UniProt, metalloproteinase- BenignNodules,extracellular Literature, 9 Symptoms space, Detection, extracellularPrediction matrix (Probable). MOGS_HUMAN Mannosyl- MOGS ENDO EndoplasmicUniProt, oligosaccharide reticulum Prediction glucosidase membrane;Single-pass type II membrane protein. MPRI_HUMAN Cation- IGF2R EPI, ENDOLungCancers, Lysosome UniProt, independent Symptoms membrane;Literature, mannose-6- Single-pass Detection phosphate type I receptormembrane protein. MRP3_HUMAN Canalicular ABCC3 EPI LungCancers Membrane;Literature, multispecific Multi-pass Detection organic membrane anionprotein. transporter 2 MUC1_HUMAN Mucin-1 MUC1 EPI LungCancers, Apicalcell UniProt, BenignNodules, membrane; Literature, Symptoms Single-passPrediction type I membrane protein. Note = Exclusively located in theapical domain of the plasma membrane of highly polarized epithelialcells. After endocytosis, internalized and recycled to the cellmembrane. Located to microvilli and to the tips of long filopodialprolusions.|Isoform 5: Secreted.|Isoform 7: Secreted.|Isoform 9:Secreted.|Mucin- 1 subunit beta: Cell membrane. Cytoplasm. Nucleus. Note= On EGF and PDGFRB stimulation, transported to the nucleus throughinteraction with CTNNB1, a process which is stimulated byphosphorylation. On HRG stimulation, colocalizes with JUP/gamma- cateninat the nucleus. MUC16_HUMAN Mucin-16 MUC16 LungCancers Cell membrane;UniProt, Single-pass Detection type I membrane protein. Secreted,extracellular space. Note = May be liberated into the extracellularspace following the phosphorylation of the intracellular C- terminuswhich induces the proteolytic cleavage and liberation of theextracellular domain. MUC4_HUMAN Mucin-4 MUC4 LungCancers, Membrane;UniProt BenignNodules Single-pass membrane protein (Potential).Secreted. Note = Isoforms lacking the Cys- rich region, EGF-like domainsand transmembrane region are secreted. Secretion occurs by splicing orproteolytic processing.|Mucin- 4 beta chain: Cell membrane; Single- passmembrane protein.|Mucin- 4 alpha chain: Secreted.|Isoform 3: Cellmembrane; Single-pass membrane protein.|Isoform 15: Secreted.MUC5B_HUMAN Mucin-5B MUC5B LungCancers, Secreted. UniProt, BenignNodulesDetection, Prediction MUCL1_HUMAN Mucin-like MUCL1 LungCancers SecretedUniProt, protein 1 (Probable). Prediction Membrane (Probable).NAMPT_HUMAN Nicotinamide NAMPT EPI LungCancers, Cytoplasm (ByLiterature, phosphoribosyl- BenignNodules, similarity). Detectiontransferase Symptoms NAPSA_HUMAN Napsin-A NAPSA Secreted LungCancersPrediction NCF4_HUMAN Neutrophil NCF4 ENDO Cytoplasm. Prediction cytosolfactor 4 NDKA_HUMAN Nucleoside NME1 Secreted LungCancers, Cytoplasm.Literature, diphosphate BenignNodules, Nucleus. Detection kinase ASymptoms Note = Cell- cycle dependent nuclear localization which can beinduced by interaction with Epstein-barr viral proteins or bydegradation of the SET complex by GzmA. NDKB_HUMAN Nucleoside NME2Secreted, BenignNodules Cytoplasm. Literature, diphosphate EPI Nucleus.Detection kinase B Note = Isoform 2 is mainly cytoplasmic and isoform 1and isoform 2 are excluded from the nucleolus. NDUS1_HUMAN NADH- NDUFS1Secreted, Symptoms Mitochondrion Prediction ubiquinone ENDO inneroxidoreductase membrane. 75 kDa subunit, mitochondrial NEBL_HUMANNebulette NEBL ENDO Prediction NEK4_HUMAN Serine/threonine- NEK4 ENDOLungCancers Nucleus Prediction protein (Probable). kinase Nek4NET1_HUMAN Netrin-1 NTN1 LungCancers, Secreted, UniProt, BenignNodulesextracellular Literature, space, Prediction extracellular matrix (Bysimilarity). NEU2_HUMAN Vasopressin- AVP LungCancers, Secreted. UniProt,neurophysin Symptoms Prediction 2- copeptin NGAL_HUMAN Neutrophil LCN2EPI LungCancers, Secreted. UniProt, gelatinase- BenignNodules,Detection, associated Symptoms Prediction lipocalin NGLY1_HUMAN Peptide-NGLY1 ENDO Cytoplasm. Detection, N(4)-(N- Prediction acetyl-beta-glucosaminyl)asparagine amidase NHRF1_HUMAN Na(+)/H(+) SLC9A3R1 EPIBenignNodules Endomembrane Detection exchange system; regulatoryPeripheral cofactor membrane NHE-RF1 protein. Cell projection,filopodium. Cell projection, ruffle. Cell projection, microvillus. Note= Colocalizes with actin in microvilli-rich apical regions of thesyncytiotrophoblast. Found in microvilli, ruffling membrane andfilopodia of HeLa cells. Present in lipid rafts of T-cells. NIBAN_HUMANProtein FAM129A EPI Cytoplasm. Literature, Niban Detection NMU_HUMANNeuromedin- NMU LungCancers Secreted. UniProt, U Prediction NRP1_HUMANNeuropilin- NRP1 LungCancers, Cell membrane; UniProt, 1 BenignNodules,Single-pass Literature, Symptoms type I Detection, membrane Predictionprotein.|Isoform 2: Secreted. ODAM_HUMAN Odontogenic ODAM LungCancersSecreted (By UniProt, ameloblast- similarity). Prediction associatedprotein OSTP_HUMAN Osteopontin SPP1 LungCancers, Secreted. UniProt,BenignNodules, Literature, Symptoms Detection, Prediction OVOS2_HUMANOvostatin OVOS2 ENDO Secreted (By UniProt, homolog 2 similarity).Prediction P5CS_HUMAN Delta-1- ALDH18A1 ENDO Mitochondrion Predictionpyrroline- inner 5- membrane. carboxylate synthase PA2GX_HUMAN Group 10PLA2G10 Symptoms Secreted. UniProt secretory phospholipase A2PAPP1_HUMAN Pappalysin- PAPPA LungCancers, Secreted. UniProt, 1BenignNodules, Literature, Symptoms Prediction PBIP1_HUMAN Pre-B-cellPBXIP1 EPI Cytoplasm, Prediction leukemia cytoskeleton. transcriptionNucleus. factor- Note = Shuttles interacting between the protein 1nucleus and the cytosol. Mainly localized in the cytoplasm, associatedwith microtubules. Detected in small amounts in the nucleus. PCBP1_HUMANPoly(rC)- PCBP1 EPI, ENDO Nucleus. Detection, binding Cytoplasm.Prediction protein 1 Note = Loosely bound in the nucleus. May shuttlebetween the nucleus and the cytoplasm. PCBP2_HUMAN Poly(rC)- PCBP2 EPINucleus. Detection, binding Cytoplasm. Prediction protein 2 Note =Loosely bound in the nucleus. May shuttle between the nucleus and thecytoplasm. PCD15_HUMAN Protocadherin- PCDH15 ENDO Cell membrane;UniProt, 15 Single-pass Detection type I membrane protein (Bysimilarity).|Isoform 3: Secreted. PCNA_HUMAN Proliferating PCNA EPILungCancers, Nucleus. Literature, cell BenignNodules, Prediction nuclearSymptoms antigen PCYOX_HUMAN Prenylcysteine PCYOX1 Secreted LungCancers,Lysosome. Detection, oxidase Symptoms Prediction 1 PDGFA_HUMAN Platelet-PDGFA LungCancers Secreted. UniProt, derived Literature, growthPrediction factor subunit A PDGFB_HUMAN Platelet- PDGFB LungCancers,Secreted. UniProt, derived BenignNodules, Literature, growth SymptomsDetection, factor Prediction subunit B PDGFD_HUMAN Platelet- PDGFDLungCancers Secreted. UniProt, derived Prediction growth factor DPDIA3_HUMAN Protein PDIA3 ENDO LungCancers Endoplasmic Detection,disulfide- reticulum Prediction isomerase lumen (By A3 similarity).Melanosome. Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. PDIA4_HUMAN Protein PDIA4 Secreted,Endoplasmic Detection, disulfide- EPI, ENDO reticulum Predictionisomerase lumen. A4 Melanosome. Note = Identified by mass spectrometryin melanosome fractions from stage I to stage IV. PDIA6_HUMAN ProteinPDIA6 Secreted, Endoplasmic Detection, disulfide- EPI, ENDO reticulumPrediction isomerase lumen (By A6 similarity). Melanosome. Note =Identified by mass spectrometry in melanosome fractions from stage I tostage IV. PECA1_HUMAN Platelet PECAM1 LungCancers, Membrane; UniProt,endothelial BenignNodules, Single-pass Literature, cell Symptoms type IDetection adhesion membrane molecule protein. PEDF_HUMAN PigmentSERPINF1 LungCancers, Secreted. UniProt, epithelium- SymptomsMelanosome. Literature, derived Note = Enriched Detection, factor instage I Prediction melanosomes. PERM_HUMAN Myeloperoxidase MPO Secreted,LungCancers, Lysosome. Literature, EPI, ENDO BenignNodules, Detection,Symptoms Prediction PERP1_HUMAN Plasma PACAP EPI, ENDO Secreted UniProt,cell- (Potential). Detection, induced Cytoplasm. Prediction residentNote = In endoplasmic (PubMed: reticulum 11350957) protein diffusegranular localization in the cytoplasm surrounding the nucleus.PGAM1_HUMAN Phosphoglycerate PGAM1 Secreted, LungCancers, Detectionmutase 1 EPI Symptoms PLAC1_HUMAN Placenta- PLAC1 LungCancers SecretedUniProt, specific (Probable). Prediction protein 1 PLACL_HUMAN Placenta-PLAC1L LungCancers Secreted UniProt, specific 1- (Potential). Predictionlike protein PLIN2_HUMAN Perilipin-2 ADFP ENDO LungCancers Membrane;Prediction Peripheral membrane protein. PLIN3_HUMAN Perilipin-3 M6PRBP1EPI Cytoplasm. Detection, Endosome Prediction membrane; Peripheralmembrane protein; Cytoplasmic side (Potential). Lipid droplet(Potential). Note = Membrane associated on endosomes. Detected in theenvelope and the core of lipid bodies and in lipid sails. PLOD1_HUMANProcollagen- PLOD1 EPI, ENDO Rough Prediction lysine, 2- endoplasmicoxoglutarate reticulum 5- membrane; dioxygenase Peripheral 1 membraneprotein; Lumenal side. PLOD2_HUMAN Procollagen- PLOD2 ENDOBenignNodules, Rough Prediction lysine, 2- Symptoms endoplasmicoxoglutarate reticulum 5- membrane; dioxygenase Peripheral 2 membraneprotein; Lumenal side. PLSL_HUMAN Plastin-2 LCP1 Secreted, LungCancersCytoplasm, Detection, EPI cytoskeleton. Prediction Cell junction. Cellprojection. Cell projection, ruffle membrane; Peripheral membraneprotein; Cytoplasmic side (By similarity). Note = Relocalizes to theimmunological synapse between peripheral blood T lymphocytes andantibody- presenting cells in response to costimulation through TCR/CD3and CD2 or CD28. Associated with the actin cytoskeleton at membraneruffles (By similarity). Relocalizes to actin-rich cell projections uponserine phosphorylation. PLUNC_HUMAN Protein PLUNC LungCancers, Secreted(By UniProt, Plunc BenignNodules similarity). Prediction Note = Found inthe nasal mucus (By similarity). Apical side of airway epithelial cells.Detected in nasal mucus (By similarity). PLXB3_HUMAN Plexin-B3 PLXNB3ENDO Membrane; UniProt, Single-pass Detection, type I Predictionmembrane protein. PLXC1_HUMAN Plexin-C1 PLXNC1 EPI Membrane; UniProt,Single-pass Detection type I membrane protein (Potential). POSTN_HUMANPeriostin POSTN Secreted, LungCancers, Secreted, UniProt, ENDOBenignNodules, extracellular Literature, Symptoms space, Detection,extracellular Prediction matrix. PPAL_HUMAN Lysosomal ACP2 EPI SymptomsLysosome UniProt, acid membrane; Prediction phosphatase Single-passmembrane protein; Lumenal side. Lysosome lumen. Note = The soluble formarises by proteolytic processing of the membrane- bound form. PPBT_HUMANAlkaline ALPL EPI LungCancers, Cell membrane; Literature, phosphatase,BenignNodules, Lipid-anchor, Detection, tissue- Symptoms GPI-anchor.Prediction nonspecific isozyme PPIB_HUMAN Peptidyl- PPIB Secreted,Endoplasmic Detection, prolyl cis- EPI, ENDO reticulum Prediction translumen. isomerase Melanosome. B Note = Identified by mass spectrometry inmelanosome fractions from stage I to stage IV. PRDX1_HUMANPeroxiredoxin- PRDX1 EPI LungCancers Cytoplasm. Detection, 1 Melanosome.Prediction Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. PRDX4_HUMAN Peroxiredoxin- PRDX4Secreted, Cytoplasm. Literature, 4 EPI, ENDO Detection, PredictionPROF1_HUMAN Profilin-1 PFN1 Secreted, LungCancers Cytoplasm, DetectionEPI cytoskeleton. PRP31_HUMAN U4/U6 PRPF31 ENDO Nucleus Prediction smallspeckle. nuclear Nucleus, Cajal ribonucleoprotein body. Prp31 Note =Predominantly found in speckles and in Cajal bodies. PRS6A_HUMAN 26SPSMC3 EPI BenignNodules Cytoplasm Detection protease (Potential).regulatory Nucleus subunit 6A (Potential). PSCA_HUMAN Prostate PSCALungCancers Cell membrane; Literature, stem cell Lipid-anchor,Prediction antigen GPI-anchor. PTGIS_HUMAN Prostacyclin PTGIS EPILungCancers, Endoplasmic UniProt, synthase BenignNodules reticulumDetection, membrane; Prediction Single-pass membrane protein. PTPA_HUMANSerine/threonine- PPP2R4 ENDO Symptoms Detection, protein Predictionphosphatase 2A activator PTPRC_HUMAN Receptor- PTPRC Secreted,LungCancers Membrane; UniProt, type EPI, ENDO Single-pass Detection,tyrosine- type I Prediction protein membrane phosphatase protein. CPTPRJ_HUMAN Receptor- PTPRJ EPI LungCancers, Membrane; UniProt, typeSymptoms Single-pass Detection, tyrosine- type I Prediction proteinmembrane phosphatase protein. eta PVR_HUMAN Poliovirus PVR SymptomsIsoform Alpha: UniProt, receptor Cell membrane; Detection, Single-passPrediction type I membrane protein.|Isoform Delta: Cell membrane;Single-pass type I membrane protein.|Isoform Beta: Secreted.|IsoformGamma: Secreted. RAB32_HUMAN Ras-related RAB32 EPI Mitochondrion.Prediction protein Rab-32 RAGE_HUMAN Advanced AGER Secreted LungCancers,Isoform 1: Cell UniProt, glycosylation BenignNodules membrane;Literature end Single-pass product- type I specific membrane receptorprotein.|Isoform 2: Secreted. RAN_HUMAN GTP- RAN Secreted, LungCancers,Nucleus. Detection, binding EPI BenignNodules Cytoplasm. Predictionnuclear Melanosome. protein Ran Note = Becomes dispersed throughout thecytoplasm during mitosis. Identified by mass spectrometry in melanosomefractions from stage I to stage IV. RAP2B_HUMAN Ras-related RAP2B EPICell membrane; Prediction protein Lipid-anchor; Rap-2b Cytoplasmic side(Potential). RAP2C_HUMAN Ras-related RAP2C EPI Cell membrane; Predictionprotein Lipid-anchor; Rap-2c Cytoplasmic side (Potential). RCN3_HUMANReticulocalbin- RCN3 EPI Symptoms Endoplasmic Prediction 3 reticulumlumen (Potential). RL24_HUMAN 60S RPL24 EPI Prediction ribosomal proteinL24 S10A1_HUMAN Protein S100A1 Symptoms Cytoplasm. Literature, S100-A1Prediction S10A6_HUMAN Protein S100A6 Secreted LungCancers NucleusLiterature, S100-A6 envelope. Detection, Cytoplasm. PredictionS10A7_HUMAN Protein S100A7 LungCancers Cytoplasm. UniProt, S100-A7Secreted. Literature, Note = Secreted Detection, by a non- Predictionclassical secretory pathway. SAA_HUMAN Serum SAA1 Symptoms Secreted.UniProt, amyloid A Literature, protein Detection, Prediction SCF_HUMANKit ligand KITLG LungCancers, Isoform 1: Cell UniProt, Symptomsmembrane; Literature Single-pass type I membrane protein (Bysimilarity). Secreted (By similarity). Note = Also exists as a secretedsoluble form (isoform 1 only) (By similarity).|Isoform 2: Cell membrane;Single-pass type I membrane protein (By similarity). Cytoplasm,cytoskeleton (By similarity). SDC1_HUMAN Syndecan-1 SDC1 LungCancers,Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms typeI Detection membrane protein. SEM3G_HUMAN Semaphorin- SEMA3G LungCancersSecreted (By UniProt, 3G similarity). Prediction SEPR_HUMAN Seprase FAPENDO Symptoms Cell membrane; UniProt, Single-pass Literature, type IIDetection membrane protein. Cell projection, lamellipodium membrane;Single-pass type II membrane protein. Cell projection, invadopodiummembrane; Single-pass type II membrane protein. Note = Found in cellsurface lamellipodia, invadopodia and on shed vesicles. SERPH_HUMANSerpin H1 SERPINH1 Secreted, LungCancers, Endoplasmic Detection, EPI,ENDO BenignNodules reticulum Prediction lumen. SFPA2_HUMAN PulmonarySFTPA2 Secreted LungCancers, Secreted, UniProt, surfactant-BenignNodules extracellular Prediction associated space, protein A2extracellular matrix. Secreted, extracellular space, surface film.SFTA1_HUMAN Pulmonary SFTPA1 Secreted LungCancers, Secreted, UniProt,surfactant- BenignNodules, extracellular Prediction associated Symptomsspace, protein A1 extracellular matrix. Secreted, extracellular space,surface film. SG3A2_HUMAN Secretoglobin SCGB3A2 LungCancers, Secreted.UniProt, family BenignNodules Prediction 3A member 2 SGPL1_HUMANSphingosine- SGPL1 ENDO Endoplasmic UniProt, 1- reticulum Predictionphosphatelyase membrane; 1 Single-pass type III membrane protein.SIAL_HUMAN Bone IBSP LungCancers Secreted. UniProt, sialoproteinLiterature, 2 Prediction SLPI_HUMAN Antileuko- SLPI LungCancers,Secreted. UniProt, proteinase BenignNodules Literature, Detection,Prediction SMD3_HUMAN Small SNRPD3 Secreted BenignNodules Nucleus.Prediction nuclear ribonucleoprotein SmD3 SMS_HUMAN Somatostatin SSTLungCancers Secreted. UniProt, Literature, Prediction SODM_HUMANSuperoxide SOD2 Secreted LungCancers, Mitochondrion Literature,dismutase BenignNodules, matrix. Detection, [Mn], Symptoms Predictionmitochondrial SORL_HUMAN Sortilin- SORL1 EPI LungCancers, Membrane;UniProt, related Symptoms Single-pass Detection receptor type I membraneprotein (Potential). SPB3_HUMAN Serpin B3 SERPINB3 LungCancers,Cytoplasm. Literature, BenignNodules Note = Seems to Detection also besecreted in plasma by cancerous cells but at a low level. SPB5_HUMANSerpin B5 SERPINB5 LungCancers Secreted, UniProt, extracellularDetection space. SPON2_HUMAN Spondin-2 SPON2 LungCancers, Secreted,UniProt, BenignNodules extracellular Prediction space, extracellularmatrix (By similarity). SPRC_HUMAN SPARC SPARC LungCancers, Secreted,UniProt, BenignNodules, extracellular Literature, Symptoms space,Detection, extracellular Prediction matrix, basement membrane. Note = Inor around the basement membrane. SRC_HUMAN Proto- SRC ENDO LungCancers,Literature oncogene BenignNodules, tyrosine- Symptoms protein kinase SrcSSRD_HUMAN Translocon- SSR4 Secreted, Endoplasmic UniProt, associatedENDO reticulum Prediction protein membrane; subunit Single-pass deltatype I membrane protein. STAT1_HUMAN Signal STAT1 EPI LungCancers,Cytoplasm. Detection transducer BenignNodules Nucleus. and Note =Translocated activator of into the transcription nucleus in 1- responseto alpha/beta IFN-gamma- induced tyrosine phosphorylation anddimerization. STAT3_HUMAN Signal STAT3 ENDO LungCancers, Cytoplasm.Prediction transducer BenignNodules, Nucleus. and Symptoms Note =Shuttles activator of between the transcription nucleus and the 3cytoplasm. Constitutive nuclear presence is independent of tyrosinephosphorylation. STC1_HUMAN Stanniocalcin-1 STC1 LungCancers, Secreted.UniProt, Symptoms Prediction STT3A_HUMAN Dolichyl- STT3A EPI SymptomsEndoplasmic Literature diphospho- reticulum oligosaccharide-- membrane;protein Multi-pass glycosyltransferase membrane subunit protein. STT3ATAGL_HUMAN Transgelin TAGLN EPI LungCancers Cytoplasm Literature,(Probable). Prediction TARA_HUMAN TRIO and TRIOBP ENDO Nucleus.Detection, F-actin- Cytoplasm, Prediction binding cytoskeleton. proteinNote = Localized to F-actin in a periodic pattern. TBA1B_HUMAN TubulinTUBA1B EPI LungCancers Detection alpha-1B chain TBB2A_HUMAN TubulinTUBB2A EPI LungCancers, Detection, beta-2A BenignNodules Predictionchain TBB3_HUMAN Tubulin TUBB3 EPI LungCancers, Detection beta-3BenignNodules chain TBB5_HUMAN Tubulin TUBE EPI LungCancers, Detectionbeta chain BenignNodules TCPA_HUMAN T-complex TCP1 EPI Cytoplasm.Prediction protein 1 subunit alpha TCPD_HUMAN T-complex CCT4 EPICytoplasm. Detection, protein 1 Melanosome. Prediction subunit Note =Identified delta by mass spectrometry in melanosome fractions from stageI to stage IV. TCPQ_HUMAN T-complex CCT8 Secreted, Cytoplasm. Predictionprotein 1 EPI subunit theta TCPZ_HUMAN T-complex CCT6A Secreted,Cytoplasm. Detection protein 1 EPI subunit zeta TDRD3_HUMAN Tudor TDRD3ENDO Cytoplasm. Prediction domain- Nucleus. containing Note =Predominantly protein 3 cytoplasmic. Associated with activelytranslating polyribosomes and with mRNA stress granules. TENA_HUMANTenascin TNC ENDO LungCancers, Secreted, UniProt, BenignNodules,extracellular Literature, Symptoms space, Detection extracellularmatrix. TENX_HUMAN Tenascin-X TNXB ENDO LungCancers, Secreted, UniProt,Symptoms extracellular Detection, space, Prediction extracellularmatrix. TERA_HUMAN Transitional VCP EPI LungCancers, Cytoplasm,Detection endoplasmic BenignNodules cytosol. reticulum Nucleus. ATPaseNote = Present in the neuronal hyaline inclusion bodies specificallyfound in motor neurons from amyotrophic lateral sclerosis patients.Present in the Lewy bodies specifically found in neurons from Parkinsondisease patients. TETN_HUMAN Tetranectin CLEC3B LungCancers Secreted.UniProt, Literature, Detection, Prediction TF_HUMAN Tissue F3LungCancers, Membrane; UniProt, factor BenignNodules, Single-passLiterature Symptoms type I membrane protein. TFR1_HUMAN Transferrin TFRCSecreted, LungCancers, Cell membrane; UniProt, receptor EPI, ENDOBenignNodules, Single-pass Literature, protein 1 Symptoms type IIDetection membrane protein. Melanosome. Note = Identified by massspectrometry in melanosome fractions from stage I to stageIV.|Transferrin receptor protein 1, serum form: Secreted. TGFA_HUMANProtransforming TGFA LungCancers, Transforming UniProt, growthBenignNodules growth factor Literature factor alpha alpha: Secreted,extracellular space.|Protransforming growth factor alpha: Cell membrane;Single-pass type I membrane protein. THAS_HUMAN Thromboxane-A TBXAS1EPI, ENDO LungCancers, Membrane; Prediction synthase BenignNodules,Multi-pass Symptoms membrane protein. THY1_HUMAN Thy-1 THY1 EPI SymptomsCell membrane; Detection, membrane Lipid-anchor, Prediction glycoproteinGPI-anchor (By similarity). TIMP1_HUMAN Metalloproteinase TIMP1LungCancers, Secreted. UniProt, inhibitor 1 BenignNodules, Literature,Symptoms Detection, Prediction TIMP3_HUMAN Metalloproteinase TIMP3LungCancers, Secreted, UniProt, inhibitor 3 BenignNodules extracellularLiterature, space, Prediction extracellular matrix. TLL1_HUMAN Tolloid-TLL1 ENDO Secreted UniProt, like protein (Probable). Prediction 1TNF12_HUMAN Tumor TNFSF12 LungCancers, Cell membrane; UniProt necrosisBenignNodules Single-pass factor type II ligand membrane superfamilyprotein.|Tumor member 12 necrosis factor ligand superfamily member 12,secreted form: Secreted. TNR6_HUMAN Tumor FAS LungCancers, Isoform 1:Cell UniProt, necrosis BenignNodules, membrane; Literature, factorSymptoms Single-pass Prediction receptor type I superfamily membranemember 6 protein.|Isoform 2: Secreted.|Isoform 3: Secreted.|Isoform 4:Secreted.|Isoform 5: Secreted.|Isoform 6: Secreted. TPIS_HUMANTriosephosphate TPI1 Secreted, Symptoms Literature, isomerase EPIDetection, Prediction TRFL_HUMAN Lactotransferrin LTF Secreted,LungCancers, Secreted. UniProt, EPI, ENDO BenignNodules, Literature,Symptoms Detection, Prediction TSP1_HUMAN Thrombospondin-1 THBS1LungCancers, Literature, BenignNodules, Detection, Symptoms PredictionTTHY_HUMAN Transthyretin TTR LungCancers, Secreted. UniProt,BenignNodules Cytoplasm. Literature, Detection, Prediction TYPH_HUMANThymidine TYMP EPI LungCancers, Literature, phosphorylase BenignNodules,Detection, Symptoms Prediction UGGG1_HUMAN UDP- UGGT1 Secreted,Endoplasmic Detection, glucose: glycoprotein ENDO reticulum Predictionglucosyltransferase lumen. 1 Endoplasmic reticulum-Golgi intermediatecompartment. UGGG2_HUMAN UDP- UGGT2 ENDO Endoplasmic Prediction glucose:glycoprotein reticulum glucosyltransferase lumen. 2 Endoplasmicreticulum-Golgi intermediate compartment. UGPA_HUMAN UTP-- UGP2 EPISymptoms Cytoplasm. Detection glucose-1- phosphate uridylyltransferaseUPAR_HUMAN Urokinase PLAUR LungCancers, Isoform 1: Cell UniProt,plasminogen BenignNodules, membrane; Literature, activator SymptomsLipid-anchor, Prediction surface GPI- receptor anchor.|Isoform 2:Secreted (Probable). UTER_HUMAN Uteroglobin SCGB1A1 LungCancers,Secreted. UniProt, BenignNodules, Literature, Symptoms Detection,Prediction VA0D1_HUMAN V-type ATP6V0D1 EPI Prediction proton ATPasesubunit d 1 VAV3_HUMAN Guanine VAV3 ENDO Prediction nucleotide exchangefactor VAV3 VEGFA_HUMAN Vascular VEGFA LungCancers, Secreted. UniProt,endothelial BenignNodules, Note = VEGF121 Literature, growth Symptoms isacidic and Prediction factor A freely secreted. VEGF165 is more basic,has heparin-binding properties and, although a signicant proportionremains cell- associated, most is freely secreted. VEGF189 is verybasic, it is cell-associated after secretion and is bound avidly byheparin and the extracellular matrix, although it may be released as asoluble form by heparin, heparinase or plasmin. VEGFC_HUMAN VascularVEGFC LungCancers, Secreted. UniProt, endothelial BenignNodulesLiterature, growth Prediction factor C VEGFD_HUMAN Vascular FIGFLungCancers Secreted. UniProt, endothelial Literature, growth Predictionfactor D VGFR1_HUMAN Vascular FLT1 LungCancers, Isoform Flt1: UniProt,endothelial BenignNodules, Cell membrane; Literature, growth SymptomsSingle-pass Detection, factor type I Prediction receptor 1 membraneprotein.|Isoform sFlt1: Secreted. VTNC_HUMAN Vitronectin VTN ENDOSymptoms Secreted, UniProt, extracellular Literature, space. Detection,Prediction VWC2_HUMAN Brorin VWC2 LungCancers Secreted, UniProt,extracellular Prediction space, extracellular matrix, basement membrane(By similarity). WNT3A_HUMAN Protein WNT3A LungCancers, Secreted,UniProt, Wnt-3a Symptoms extracellular Prediction space, extracellularmatrix. WT1_HUMAN Wilms WT1 LungCancers, Nucleus. Literature, tumorBenignNodules, Cytoplasm (By Prediction protein Symptoms similarity).Note = Shuttles between nucleus and cytoplasm (By similarity).|Isoform1: Nucleus speckle.|Isoform 4: Nucleus, nucleoplasm. ZA2G_HUMANZinc-alpha- AZGP1 LungCancers, Secreted. UniProt, 2- SymptomsLiterature, glycoprotein Detection, Prediction ZG16B_HUMAN Zymogen ZG16BLungCancers Secreted UniProt, granule (Potential). Prediction protein 16homolog B

SRM Assay

SRM assays for 388 targeted proteins were developed based on syntheticpeptides, using a protocol similar to those described in the literature(Lange, Picotti et al. 2008, Picotti, Rinner et al. 2010, Huttenhain,Soste et al. 2012). Up to five SRM suitable peptides per protein wereidentified from public sources such as the PeptideAtlas, Human PlasmaProteome Database or by proteotypic prediction tools (Mallick, Schirleet al. 2007) and synthesized. SRM triggered MS/MS spectra were collectedon an ABSciex 5500 QTrap for both doubly and triply charged precursorions. The obtained MS/MS spectra were assigned to individual peptidesusing MASCOT (cutoff score ≧15) (Perkins, Pappin et al. 1999). Up tofour transitions per precursor ion were selected for optimization. Theresulting corresponding optimal retention time, declustering potentialand collision energy were assembled for all transitions. Optimaltransitions were measured on a mixture of all synthetic peptides, apooled sample of benign patients and a pooled sample of cancer patients.Transitions were analyzed in batches, each containing up to 1750transitions. Both biological samples were immuno-depleted and digestedby trypsin. All three samples were analyzed on an ABSciex 5500 QTrapcoupled with a reversed-phase (RP) high-performance liquidchromatography (HPLC) system. The obtained SRM data were manuallyreviewed to select the two best peptides per protein and the two besttransitions per peptide. Transitions having interference with othertransitions were not selected. Ratios between intensities of the twobest transitions of peptides in the synthetic peptide mixture were alsoused to assess the specificity of the transitions in the biologicalsamples. The intensity ratio was considered as an important metricdefining the SRM assays. The complete transition table is shown below inTable 2.

Lengthy table referenced here US20170212120A1-20170727-T00001 Pleaserefer to the end of the specification for access instructions.

Exemplary Protein Detection

The following 164 proteins and their peptides were detectedsimultaneously in a large-scale experiment of 158 samples using theMS-LC-SRM-MS system described herein.

TABLE 3 SEQ ID Protein Peptide Protein Peptide NO: Detection Detection1433E_HUMAN EDLVYQAK 7 16 16 1433E_HUMAN IISSIEQK 9 16 0 1433T_HUMANAVTEQGAELSNEER 16 127 0 1433T_HUMAN TAFDEAIAELDTLNEDSYK 19 127 1271433Z_HUMAN FLIPNASQAESK 21 157 157 1433Z_HUMAN SVTEQGAELSNEER 23 157 06PGD_HUMAN AGQAVDDFIEK 25 90 0 6PGD_HUMAN LVPLLDTGDIIIDGGNSEYR 27 90 90A1AG1_HUMAN WFYIASAFR 32 157 0 A1AG1_HUMAN YVGGQEHFAHLLILR 33 157 157ABCD1_HUMAN DAGIALLSITHRPSLWK 34 9 0 ABCD1_HUMAN GLQAPAGEPTQEASGVAAAK 369 0 ABCD1_HUMAN NLLTAAADAIER 37 9 9 ADML_HUMAN LAHQIYQFTDK 44 27 27ADML_HUMAN SPEDSSPDAAR 45 27 0 AIFM1_HUMAN ELWFSDDPNVTK 53 158 158AIFM1_HUMAN GVIFYLR 54 158 0 ALDOA_HUMAN ADDGRPFPQVIK 57 158 141ALDOA_HUMAN ALQASALK 58 158 17 AMPN_HUMAN ALEQALEK 62 158 158 AMPN_HUMANDHSAIPVINR 63 158 0 APOA1_HUMAN AKPALEDLR 78 158 158 APOA1_HUMANATEHLSTLSEK 79 158 0 APOE_HUMAN AATVGSLAGQPLQER 82 158 158 APOE_HUMANLGPLVEQGR 87 158 0 BGH3_HUMAN LTLLAPLNSVFK 139 158 0 BGH3_HUMANSPYQLVLQHSR 140 158 158 BST1_HUMAN GEGTSAHLR 149 157 0 BST1_HUMANGFFADYEIPNLQK 150 157 157 C163A_HUMAN INPASLDK 153 158 11 C163A_HUMANLEVFYNGAWGTVGK 154 158 49 C163A_HUMAN TSYQVYSK 155 158 98 CALU_HUMANEQFVEFR 172 120 120 CALU_HUMAN TFDQLTPEESK 174 120 0 CATB_HUMAN LPASFDAR188 62 62 CATB_HUMAN TDQYWEK 190 62 0 CATG_HUMAN NVNPVALPR 192 14 0CATG_HUMAN SSGVPPEVFTR 193 14 14 CBPB2_HUMAN DTGTYGFLLPER 198 158 158CBPB2_HUMAN EAFAAVSK 199 158 0 CD14_HUMAN ATVNPSAPR 207 158 0 CD14_HUMANSWLAELQQWLKPGLK 214 158 158 CD44_HUMAN FAGVFHVEK 227 158 158 CD44_HUMANYGFIEGHVVIPR 231 158 0 CD59_HUMAN AGLQVYNK 232 156 156 CD59_HUMANTVLLLVTPFLAAAWSLHP 233 156 0 CDCP1_HUMAN EEGVFTVTPDTK 239 157 0CDCP1_HUMAN LSLVLVPAQK 241 157 157 CEAM8_HUMAN LFIPNITTK 256 79 79CEAM8_HUMAN TLTLLSVTR 257 79 0 CERU_HUMAN GAYPLSIEPIGVR 258 158 0CERU_HUMAN GPEEEHLGILGPVIWAEVGDTIR 259 158 158 CERU_HUMANNNEGTYYSPNYNPQSR 261 158 0 CH10_HUMAN GGEIQPVSVK 265 158 0 CH10_HUMANVLLPEYGGTK 266 158 158 CLIC1_HUMAN FSAYIK 288 137 8 CLIC1_HUMANLAALNPESNTAGLDIFAK 290 137 129 CLIC1_HUMAN NSNPALNDNLEK 291 137 0CLUS_HUMAN ASSIIDELFQDR 293 158 0 CLUS_HUMAN EIQNAVNGVK 294 158 158CNTN1_HUMAN AHSDGGDGVVSQVK 303 158 157 CNTN1_HUMAN DGEYVVEVR 304 158 1CO6A3_HUMAN IGDLHPQIVNLLK 319 158 0 CO6A3_HUMAN VAVVQYSDR 321 158 158CO6A3_HUMAN WYYDPNTK 322 158 0 COF1_HUMAN EILVGDVGQTVDDPYATFVK 328 127 0COF1_HUMAN LGGSAVISLEGKPL 329 127 0 COF1_HUMAN YALYDATYETK 330 127 127COIA1_HUMAN AVGLAGTFR 332 158 37 COIA1_HUMAN TEAPSATGQASSLLGGR 335 158121 CRP_HUMAN APLTKPLK 341 153 21 CRP_HUMAN ESDTSYVSLK 342 153 132CRP_HUMAN YEVQGEVFTKPQLWP 343 153 0 CSF1_HUMAN FNSVPLTDTGHER 351 134 113CSF1_HUMAN ISSLRPQGLSNPSTLSAQPQLSR 352 134 21 CYTB_HUMAN SQLEEK 372 1000 CYTB_HUMAN SQVVAGTNYFIK 373 100 100 DESP_HUMAN YGDGIQLTR 384 131 131DMKN_HUMAN QVPGFGVADALGNR 395 128 0 DMKN_HUMAN VSEALGQGTR 397 128 128DSG2_HUMAN GQIIGNFQAFDEDTGLPAHAR 404 158 1 DSG2_HUMAN ILDVNDNIPVVENK 405158 157 EF1A1_HUMAN IGGIGTVPVGR 423 158 158 EF1A1_HUMAN QTVAVGVIK 426158 0 EF2_HUMAN FSVSPVVR 439 125 125 EF2_HUMAN GVQYLNEIK 441 125 0ENOA_HUMAN AVEHINK 452 156 0 ENOA_HUMAN YISPDQLADLYK 455 156 156ENOA_HUMAN YNQLLR 456 156 0 ENPL_HUMAN SGTSEFLNK 469 158 1 ENPL_HUMANSGYLLPDTK 470 158 157 EPHB6_HUMAN RPHFDQLVAAFDK 472 157 0 EPHB6_HUMANWAAPEVIAHGK 476 157 157 ERBB3_HUMAN GESIEPLDPSEK 483 105 0 ERBB3_HUMANLAEVPDLLEK 484 105 105 EREG_HUMAN VAQVSITK 487 115 115 EREG_HUMANVTSGDPELPQV 488 115 0 ERO1A_HUMAN AVLQVVTK 489 121 0 ERO1A_HUMANLLESDYFR 491 121 0 ERO1A_HUMAN NLLQNIH 492 121 0 ERO1A_HUMANVLPFFERPDFQLFTGNK 493 121 121 F10A1_HUMAN AIDLFTDAIK 501 35 0F10A1_HUMAN LQKPNAAIR 503 35 35 FAM3C_HUMAN GINVALANGK 505 97 88FAM3C_HUMAN SALDTAAR 507 97 9 FAM3C_HUMAN TGEVLDTK 509 97 0 FCGR1_HUMANHLEEELK 517 39 0 FCGR1_HUMAN VFTEGEPLALR 519 39 39 FIBA_HUMANGGSTSYGTGSETESPR 554 147 108 FIBA_HUMAN NSLFEYQK 556 147 39 FINC_HUMANSYTITGLQPGTDYK 561 154 135 FINC_HUMAN VPGTSTSATLTGLTR 562 154 19FKB11_HUMAN ANYWLK 565 23 0 FKB11_HUMAN DPLVIELGQK 566 23 23 FOLH1_HUMANGVILYSDPADYFAPGVK 569 138 0 FOLH1_HUMAN LGSGNDFEVFFQR 570 138 138FRIL_HUMAN DDVALEGVSHFFR 594 151 0 FRIL_HUMAN LGGPEAGLGEYLFER 596 151151 G3P_HUMAN GALQNIIPASTGAAK 599 150 149 G3P_HUMAN LISWYDNEFGYSNR 600150 1 G6PD_HUMAN DGLLPENTFIVGYAR 603 43 43 G6PD_HUMAN GGYFDEFGIIR 604 430 G6PI_HUMAN AVLHVALR 608 39 6 G6PI_HUMAN TLAQLNPESSLFIIASK 610 39 33GDIR2_HUMAN DIVSGLK 629 158 158 GDIR2_HUMAN LNYKPPPQK 630 158 0GELS_HUMAN AQPVQVAEGSEPDGFWEALGGK 634 158 0 GELS_HUMAN TASDFITK 637 158158 GGH_HUMAN NLDGISHAPNAVK 640 158 158 GGH_HUMAN YYIAASYVK 643 158 0GRP78_HUMAN TWNDPSVQQDIK 664 158 90 GRP78_HUMAN VYEGERPLTK 665 158 68GSLG1_HUMAN IIIQESALDYR 666 158 158 GSLG1_HUMAN LDPALQDK 667 158 0GSLG1_HUMAN LIAQDYK 668 158 0 GSLG1_HUMAN NDINILK 669 158 0 GSTP1_HUMANALPGQLKPFETLLSQNQGGK 672 123 123 GSTP1_HUMAN YISLIYTNYEAGK 675 123 0HPSE_HUMAN LPYPFSNK 714 49 0 HPSE_HUMAN SVQLNGLTLK 715 49 49 HPT_HUMANVGYVSGWGR 719 158 0 HPT_HUMAN VTSIQDWVQK 720 158 158 HS90A_HUMANSLTNDWEDHLAVK 724 32 32 HS90B_HUMAN ADHGEPIGR 726 121 0 HS90B_HUMANIDIIPNPQER 728 121 121 HS90B_HUMAN NPDDITQEEYGEFYK 730 121 0 HSPB1_HUMANDGVVEITGK 732 30 0 HSPB1_HUMAN GPSWDPFR 733 30 30 HTRA1_HUMANLHRPPVIVLQR 741 40 40 HTRA1_HUMAN LPVLLLGR 742 40 0 HTRA1_HUMANVTAGISFAIPSDK 744 40 0 HXK1_HUMAN FLLSESGSGK 747 117 17 HXK1_HUMANLVDEYSLNAGK 749 117 47 HXK1_HUMAN SANLVAATLGAILNR 750 117 53 HYOU1_HUMANFPEHELTFDPQR 757 156 0 HYOU1_HUMAN LPATEKPVLLSK 760 156 156 IBP2_HUMANAEVLFR 762 158 0 IBP2_HUMAN ELAVFR 763 158 158 IBP2_HUMAN LIQGAPTIR 765158 0 IBP3_HUMAN FHPLHSK 768 158 0 IBP3_HUMAN FLNVLSPR 769 158 0IBP3_HUMAN YGQPLPGYTTK 771 158 158 ICAM1_HUMAN ASVSVTAEDEGTQR 772 114 0ICAM1_HUMAN VELAPLPSWQPVGK 776 114 114 ICAM3_HUMAN IALETSLSK 780 158 0ICAM3_HUMAN TFVLPVTPPR 783 158 158 IF4A1_HUMAN GYDVIAQAQSGTGK 792 58 0IF4A1_HUMAN VLITTDLLAR 796 58 58 IGF1_HUMAN EGTEASLQIR 797 40 0IGF1_HUMAN ISSLPTQLFK 798 40 40 IL18_HUMAN SDIIFFQR 807 45 45 IL18_HUMANSVPGHDNK 808 45 0 ILEU_HUMAN EATTNAPFR 824 88 13 ILEU_HUMANTYNFLPEFLVSTQK 828 88 75 ILK_HUMAN HSGIDFK 830 90 15 ILK_HUMAN QLNFLTK832 90 75 ILK_HUMAN WQGNDIVVK 833 90 0 INHBA_HUMAN AEVWLFLK 834 32 0INHBA_HUMAN EGSDLSVVER 835 32 32 ISLR_HUMAN ALPGTPVASSQPR 839 158 0ISLR_HUMAN EVPLLQSLWLAHNEIR 840 158 0 ISLR_HUMAN LPGLPEGAFR 841 158 158ITA5_HUMAN SLQWFGATVR 846 114 114 ITA5_HUMAN SSASSGPQILK 847 114 0K1C18_HUMAN LASYLDR 861 72 0 K1C18_HUMAN LQLETEIEALK 862 72 72K1C18_HUMAN VVSETNDTK 863 72 0 K1C19_HUMAN FGAQLAHIQALISGIEAQLGDVR 865158 158 K1C19_HUMAN FGPGVAFR 866 158 0 KIT_HUMAN QATLTISSAR 876 158 158KIT_HUMAN YVSELHLTR 878 158 0 KLK14_HUMAN VLGSGTWPSAPK 889 27 27KLK14_HUMAN VSGWGTISSPIAR 890 27 0 KPYM_HUMAN APIIAVTR 899 158 13KPYM_HUMAN LDIDSPPITAR 903 158 145 LAMB2_HUMAN IQGTLQPHAR 910 69 0LAMB2_HUMAN SLADVDAILAR 911 69 31 LAMB2_HUMAN VLELSIPASAEQIQHLAGAIAER913 69 38 LDHA_HUMAN FIIPNVVK 915 157 0 LDHA_HUMAN LVIITAGAR 917 157 157LDHB_HUMAN FIIPQIVK 920 158 157 LDHB_HUMAN GLTSVINQK 921 158 1LEG1_HUMAN GEVAPDAK 925 146 0 LEG1_HUMAN LPDGYEFK 926 146 0 LEG1_HUMANSFVLNLGK 927 146 146 LG3BP_HUMAN ASHEEVEGLVEK 938 158 158 LG3BP_HUMANVEIFYR 941 158 0 LG3BP_HUMAN YSSDYFQAPSDYR 942 158 0 LRP1_HUMANTVLWPNGLSLDIPAGR 959 158 158 LRP1_HUMAN VFFTDYGQIPK 960 158 0 LUM_HUMANNIPTVNENLENYYLEVNQLEK 962 158 158 LUM_HUMAN SLEDLQLTHNK 964 158 0LYOX_HUMAN HWFQAGYSTSR 975 121 0 LYOX_HUMAN TPILLIR 977 121 121MASP1_HUMAN APGELEHGLITFSTR 991 158 151 MASP1_HUMAN TGVITSPDFPNPYPK 994158 7 MDHC_HUMAN LGVTANDVK 997 130 130 MDHC_HUMANVLVTGAAGQIAYSLLYSIGNGSVFGK 999 130 0 MDHM_HUMAN VDFPQDQLTALTGR 1002 158158 MDHM_HUMAN VSSFEEK 1004 158 0 MMP12_HUMAN FLLILLLQATASGALPLNSSTSLEK1021 158 158 MMP12_HUMAN GIQSLYGDPK 1022 158 0 MMP12_HUMAN IDAVFYSK 1023158 0 MMP2_HUMAN AFQVWSDVTPLR 1031 153 152 MMP2_HUMANIIGYTPDLDPETVDDAFAR 1033 153 1 MMP7_HUMAN LSQDDIK 1042 102 102MMP7_HUMAN NANSLEAK 1043 102 0 MMP9_HUMAN AFALWSAVTPLTFTR 1044 158 50MMP9_HUMAN FQTFEGDLK 1046 158 108 MMP9_HUMAN SLGPALLLLQK 1048 158 0MPRI_HUMAN GHQAFDVGQPR 1055 158 23 MPRI_HUMAN TYHSVGDSVLR 1056 158 4MPRI_HUMAN VPIDGPPIDIGR 1057 158 131 NCF4_HUMAN AEALFDFTGNSK 1095 138 43NCF4_HUMAN DAEGDLVR 1096 138 0 NCF4_HUMAN DIAVEEDLSSTPLLK 1097 138 0NCF4_HUMAN GATGIFPLSFVK 1098 138 95 NDKB_HUMAN DRPFFPGLVK 1105 24 0NDKB_HUMAN NIIHGSDSVK 1107 24 24 NRP1_HUMAN FVSDYETHGAGFSIR 1149 158 0NRP1_HUMAN FVTAVGTQGAISK 1150 158 158 NRP1_HUMAN SFEGNNNYDTPELR 1152 1580 OSTP_HUMAN AIPVAQDLNAPSDWDSR 1156 108 108 OSTP_HUMANDSYETSQLDDQSAETHSHK 1157 108 0 OSTP_HUMAN YPDAVATWLNPDPSQK 1160 108 0PCBP2_HUMAN IANPVEGSTDR 1189 52 0 PCBP2_HUMAN IITLAGPTNAIFK 1190 52 52PCYOX_HUMAN IAIIGAGIGGTSAAYYLR 1207 37 0 PCYOX_HUMAN IFSQETLTK 1208 3737 PCYOX_HUMAN TLLETLQK 1209 37 0 PDGFB_HUMAN SFDDLQR 1216 111 99PDGFB_HUMAN SHSGGELESLAR 1217 111 12 PDIA3_HUMAN ELSDFISYLQR 1225 129129 PDIA3_HUMAN SEPIPESNDGPVK 1227 129 0 PDIA4_HUMAN FDVSGYPTIK 1231 8181 PDIA4_HUMAN FHHTFSTEIAK 1232 81 0 PECA1_HUMAN SELVTVTESFSTPK 1241 770 PECA1_HUMAN STESYFIPEVR 1242 77 77 PEDF_HUMAN LQSLFDSPDFSK 1246 158 0PEDF_HUMAN TVQAVLTVPK 1248 158 158 PGAM1_HUMAN HGESAWNLENR 1259 14 14PLIN2_HUMAN DAVTTTVTGAK 1264 138 0 PLIN2_HUMAN EVSDSLLTSSK 1265 138 138PLSL_HUMAN IGNFSTDIK 1284 158 0 PLSL_HUMAN ISFDEFIK 1285 158 158PLX63_HUMAN ELPVPIYVTQGEAQR 1294 77 0 PLX63_HUMAN GPVDAVTGK 1296 77 77PLXC1_HUMAN FWVNILK 1299 158 0 PLXC1_HUMAN LNTIGHYEISNGSTIK 1300 158 158POSTN_HUMAN GFEPGVTNILK 1302 158 158 POSTN_HUMAN IIDGVPVEITEK 1303 158 0POSTN_HUMAN IIHGNQIATNGVVHVIDR 1304 158 0 PPIB_HUMAN VIFGLFGK 1319 158 0PPIB_HUMAN VYFDLR 1320 158 158 PRDX1_HUMAN IGHPAPNFK 1325 158 116PRDX1_HUMAN QITVNDLPVGR 1328 158 42 PROF1_HUMAN STGGAPTFNVTVTK 1338 158157 PROF1_HUMAN TFVNITPAEVGVLVGK 1339 158 1 PRS6A_HUMAN VDILDPALLR 134913 13 PTGIS_HUMAN DPEIYTDPEVFK 1352 158 0 PTGIS_HUMAN LLLFPFLSPQR 1357158 158 PTPA_HUMAN FGSLLPIHPVTSG 1361 103 103 PTPA_HUMANTGPFAEHSNQLWNISAVPSWSK 1363 103 0 PTPA_HUMAN VDDQIAIVFK 1364 103 0PTPA_HUMAN WIDETPPVDQPSR 1365 103 0 PTPRJ_HUMAN AVSISPTNVILTWK 1372 1580 PTPRJ_HUMAN VITEPIPVSDLR 1374 158 158 PVR_HUMAN SVDIWLR 1379 158 158PVR_HUMAN VLAKPQNTAEVQK 1380 158 0 RAB32_HUMAN VHLPNGSPIPAVLLANK 1384 220 RAB32_HUMAN VLVIGELGVGK 1385 22 22 RAN_HUMAN FNVWDTAGQEK 1391 116 2RAN_HUMAN LVLVGDGGTGK 1392 116 114 RAN_HUMAN NVPNWHR 1393 116 0RAP2B_HUMAN EVSYGEGK 1395 145 0 RAP2B_HUMAN VDLEGER 1397 145 145S10A1_HUMAN DVDAVDK 1408 128 128 S10A1_HUMAN ELLQTELSGFLDAQK 1409 128 0S10A6_HUMAN ELTIGSK 1411 154 154 S10A6_HUMAN LQDAEIAR 1412 154 0SAA_HUMAN EANYIGSDK 1414 143 0 SAA_HUMAN SFFSFLGEAFDGAR 1416 143 143SCF_HUMAN LFTPEEFFR 1418 143 143 SCF_HUMAN LVANLPK 1419 143 0SEM3G_HUMAN DYPDEVLQFAR 1426 155 0 SEM3G_HUMAN LFLGGLDALYSLR 1428 155155 SIAL_HUMAN AYEDEYSYFK 1449 19 19 SIAL_HUMAN TTSPPFGK 1452 19 0SODM_HUMAN GDVTAQIALQPALK 1460 154 151 SODM_HUMAN NVRPDYLK 1462 154 3SPON2_HUMAN WSQTAFPK 1478 63 0 SPON2_HUMAN YSITFTGK 1479 63 63STAT1_HUMAN TELISVSEVHPSR 1494 38 29 STAT1_HUMAN YTYEHDPITK 1496 38 9TBA1B_HUMAN AVFVDLEPTVIDEVR 1519 119 119 TBA1B_HUMAN EIIDLVLDR 1520 1190 TBB3_HUMAN ISVYYNEASSHK 1533 158 158 TBB3_HUMAN YLTVATVFR 1535 158 0TCPA_HUMAN IHPTSVISGYR 1540 158 4 TCPA_HUMAN SSLGPVGLDK 1542 158 154TCPQ_HUMAN DIDEVSSLLR 1550 48 0 TCPQ_HUMAN NVGLDIEAEVPAVK 1553 48 48TCPZ_HUMAN GIDPFSLDALSK 1557 6 6 TCPZ_HUMAN GLVLDHGAR 1558 6 0TENA_HUMAN GLEPGQEYNVLLTAEK 1570 140 140 TENA_HUMANTVSGNTVEYALTDLEPATEYTLR 1572 140 0 TENX_HUMAN DAQGQPQAVPVSGDLR 1574 158158 TENX_HUMAN YEVTVVSVR 1578 158 0 TERA_HUMAN GILLYGPPGTGK 1579 106 94TERA_HUMAN LDQLIYIPLPDEK 1582 106 12 TETN_HUMAN GGTLSTPQTGSENDALYEYLR1588 158 118 TETN_HUMAN LDTLAQEVALLK 1589 158 40 TFR1_HUMAN LTVSNVLK1598 157 0 TFR1_HUMAN SSGLPNIPVQTISR 1600 157 157 TIMP1_HUMANGFQALGDAADIR 1610 151 151 TIMP1_HUMAN SEEFLIAGK 1611 151 0 TNF12_HUMANAAPFLTYFGLFQVH 1621 156 156 TNF12_HUMAN INSSSPLR 1622 156 0 TPIS_HUMANVVFEQTK 1634 157 157 TPIS_HUMAN VVLAYEPVWAIGTGK 1635 157 0 TRFL_HUMANFQLFGSPSGQK 1637 48 22 TRFL_HUMAN LRPVAAEVYGTER 1638 48 4 TRFL_HUMANVPSHAVVAR 1639 48 5 TRFL_HUMAN YYGYTGAFR 1640 48 17 TSP1_HUMAN GFLLLASLR1644 158 6 TSP1_HUMAN GTSQNDPNWVVR 1645 158 152 TTHY_HUMANTSESGELHGLTTEEEFVEGIYK 1646 27 27 TTHY_HUMAN VEIDTK 1647 27 0 TYPH_HUMANALQEALVLSDR 1648 59 0 TYPH_HUMAN TLVGVGASLGLR 1651 59 59 UGGG1_HUMANDLSQNFPTK 1653 58 58 UGGG1_HUMAN FTILDSQGK 1654 58 0 UGPA_HUMANLVEIAQVPK 1669 97 97 UGPA_HUMAN NENTFLDLTVQQIEHLNK 1670 97 0 VA0D1_HUMANLLFEGAGSNPGDK 1679 13 13 VA0D1_HUMAN NVADYYPEYK 1681 13 0 VEGFC_HUMANDLEEQLR 1689 21 21 VEGFC_HUMAN EAPAAAAAFESGLDLSDAEPDAGEA 1690 21 0TAYASK VEGFC_HUMAN FAAAHYNTEILK 1692 21 0 VEGFC_HUMAN NQPLNPGK 1693 21 0VTNC_HUMAN AVRPGYPK 1705 145 0 VTNC_HUMAN DVWGIEGPIDAAFTR 1707 145 145ZA2G_HUMAN EIPAWVPFDPAAQITK 1724 158 158 ZA2G_HUMAN WEAEPVYVQR 1725 1580

Exemplary Biomarker Protein

The following 36 proteins were identified as biomarker candidates in alarge-scale experiment of 72 lung cancer samples and 71 benign lungnodule samples using the MS-LC-SRM-MS system described herein.

TABLE 4 Co- Co- efficient efficient Predicted Protein Official Cooper-Co- SEQ (Discovery) (Final) Concen- (Unit- Gene ative Partial efficientID alpha = alpha = Tissue traion Category Prot) Name Score AUC CVTransition NO: 36.16 26.25 Candiate (ng/ml) Classifier TSP1_ THBS1 1.80.25 0.24 GFLLLASLR_ 1644 0.53 0.44 510 HUMAN 495.31_ 559.40 ClassifierCOIA1_ COL18A1 3.7 0.16 0.25 AVGLAGTFR_ 332 -1.56 -0.91 35 HUMAN 446.26_721.40 Classifier ISLR_ ISLR 1.4 0.32 0.25 ALPGTPVAS 839 1.40 0.83 —HUMAN SQPR_ 640.85_ 841.50 Classifier TETN_ CLEC3B 2.5 0.26 0.26LDTLAQEVAL 1589 -1.79 -1.02 58000 HUMAN LK_ 657.39_ 330.20 ClassifierFRIL_ FTL 2.8 0.31 0.26 LFFPAGLG 596 0.36 0.14 Secreted 12 HUMAN EYLFER_Epi, 804.40_ Endo 913.40 Classifier GRP78_ HSPA5 1.4 0.27 0.27 TWNDPSVQ664 1.41 0.55 Secreted 100 HUMAN QDIK_ Epi, 715.85_ Endo 260.20Classifier ALDOA_ ALDOQ 1.3 0.26 0.28 ALQASLK_ 58 -0.80 -0.26 Secreted250 HUMAN 401.25_ Epi, 617.40 Classifier BGH3_ TGFBI 1.8 0.21 0.28LTLLAPLNSV 139 1.73 0.54 Epi 1.40 HUMAN FK_ 658.40_ 804.50 ClassifierLG3BP_ LGALS3BP 4.3 0.29 0.29 VEIFYR_ 941 -0.58 -0.21 Secreted 440 HUMAN413.73_ 598.30 Classifier LRP1_ LRP1 4.0 0.13 0.32 TVLWPNGLS 959 -1.59-0.83 Epi 20 HUMAN LDIPAGR_ 855.00_ 400.20 Classifier FIBA_ FGA 1.1 0.310.35 NSLFEYQK_ 556 0.31 0.13 130000 HUMAN 514.76_ 714.30 ClassifierPRDX1_ PRDX1 1.5 0.32 0.37 QITVNDLPVG_ 1328 -0.34 -0.26 Epi 60 HUMANR_606.30_ 428.30 Classifier GSLG1_ GLG1 1.2 0.34 0.45 IIIQESALDYR_ 666-0.70 -0.44 Epi, — HUMAN 660.86_ Endo 338.20 Robust KIT_ KIT 1.4 0.330.46 8.2 HUMAN Robust CD14_ CD14 4.0 0.33 0.48 Epi 420 HUMAN RobustEF1A1_ EF1A1 1.2 0.32 0.56 Secreted, 61 HUMAN Epi Robust TENX_ TNXB 1.10.30 0.56 Endo 70 HUMAN Robust AIFM1_ AIFM1 1.4 0.32 0.70 Epi, 1.4 HUMANEndo Robust GGH_ GGH 1.3 0.32 0.81 250 HUMAN Robust IBP3_ IGFBP3 3.40.32 1.82 5700 HUMAN Robust ENPL_ HSP90B1 1.1 0.29 5.90 Secreted, 88HUMAN Epi, Endo Non- ERO1A_ ERO1L 6.2 Secreted, — Robust HUMAN Epi, EndoNon- 6PGD_ PGD 4.3 Epi, 29 Robust HUMAN Endo Non- ICAM1_ ICAM1_ 3.9 71Robust HUMAN Non- PTPA_ PPP2R4 2.1 Endo 3.3 Robust HUMAN Non- NCF4_ NCF42.0 Endo — Robust HUMAN Non- SEM3G_ SEM3G 1.9 — Robust HUMAN Non- 1433T_YWHAQ 1.5 Epi 180 Robust HUMAN Non- RAP2B_ RAP2B 1.5 Epi — Robust HUMANNon- MMP9_ MMP9 1.4 28 Robust HUMAN Non- FOLH1_ FOLH1 1.3 — Robust HUMANNon- GSTP1_ GSTP1 1.3 Endo 32 Robust HUMAN Non- EF2_ EEF2 1.3 Secreted,30 Robust HUMAN Epi Non- RAN_ RAN 1.2 Secreted, 4.6 Robust HUMAN EpiNon- SODM_ SOD2 1.2 Secreted 7.1 Robust HUMAN Non- DSG2_ DSG2 1.1 Endo2.7 Robust HUMAN

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20170212120A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A composition comprising isolated synthetic peptides said compositioncomprising ALQASALK (SEQ ID NO: 58), GFLLLASLR (SEQ ID NO: 1644),LGGPEAGLGEYLFER (SEQ ID NO: 596), YVSELHLTR (SEQ ID NO: 878), andYYIAASYVK (SEQ ID NO: 643).
 2. A composition comprising at least 10isolated synthetic peptides said composition comprising at least 2isolated synthetic peptides of at least 5 proteins selected from LRP1,BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA,KIT, GGH or GSLG1, and wherein the at least two isolated syntheticpeptides are selected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQID NO: 58), QLLLTADDR (SEQ ID NO: 60), ADHHATNGVVHLIDK (SEQ ID NO: 136),DILATNGVIHYIDELLIPDSAK (SEQ ID NO: 137), LTLLAPLNSVFK (SEQ ID NO: 139),SPYQLVLQHSR (SEQ ID NO: 140), ADDILASPPR (SEQ ID NO: 331), AVGLAGTFR(SEQ ID NO: 332), TEAPSATGQASSLLGGR (SEQ ID NO: 335), GGSTSYGTGSETESPR(SEQ ID NO 554), NSLFEYQK (SEQ ID NO: 556), TVIGPDGHK (SEQ ID NO: 557),VQHIQLLQK (SEQ ID NO: 558), ALFQDIK (SEQ ID NO: 593), DDVALEGVSHFFR (SEQID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER (SEQ ID NO:596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598),LYGSAGPPPTGEEDTAEK (SEQ ID NO: 663), TWNDPSVQQDIK (SEQ ID NO: 664),VYEGERPLTK (SEQ ID NO: 665), IIIQESALDYR (SEQ ID NO: 666), LDPALQDK (SEQID NO: 667), LIAQDYK (SEQ ID NO: 668), NDINILK (SEQ ID NO: 669),VAELSSDDFHLDR (SEQ ID NO: 670), ALPGTPVASSQPR (SEQ ID NO: 839),EVPLLQSLWLAHNEIR (SEQ ID NO: 840), LPGLPEGAFR (SEQ ID NO: 841),TVAAGALASLSHLK (SEQ ID NO: 843), ASHEEVEGLVEK (SEQ ID NO: 938),STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQID NO: 942), AALSGANVLTLIEK (SEQ ID NO: 958), TVLWPNGLSLDIPAGR (SEQ IDNO: 959), VFFTDYGQIPK (SEQ ID NO: 960), ADEGISFR (SEQ ID NO: 1321),ATAVMPDGQFK (SEQ ID NO: 1322), DISLSDYK (SEQ ID NO: 1323), GLFIIDDK (SEQID NO: 1324), IGHPAPNFK (SEQ ID NO: 1325), LVQAFQFTDK (SEQ ID NO: 1326),QGGLGPMNIPLVSDPK (SEQ ID NO: 1327), QITVNDLPVGR (SEQ ID NO: 1328),SVDETLR (SEQ ID NO: 1329), TIAQDYGVLK (SEQ ID NO: 1330),GGTLSTPQTGSENDALYEYLR (SEQ ID NO: 1588), LDTLAQEVALLK (SEQ ID NO: 1589),NWETEITAQPDGGK (SEQ ID NO: 1590), FQDLVDAVR (SEQ ID NO: 1641),FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643),GFLLLASLR (SEQ ID NO: 1644), YVSELHLTR (SEQ ID NO: 878), YYIAASYVK (SEQID NO: 643) or GTSQNDPNWVVR (SEQ ID NO: 1645).
 3. The composition ofclaim 2, wherein at least one of the isolated synthetic peptides isisotopically labeled.
 4. The composition of claim 2, wherein themeasured quantity of each of the at least 2 isolated synthetic peptidesof at least 5 proteins is known.
 5. The composition of claim 2, furthercomprising one or more polar solvents.
 6. The composition of claim 2,further comprising an additional five isolated synthetic peptides of atleast one protein selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA,GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT, GGH or GSLG1.
 7. Thecomposition of claim 2, further comprising an additional five isolatedsynthetic peptides of at least one protein selected from APOE, BASP1,CD14, FOXA2 or HSPB1.
 8. A composition comprising at least 25 isolatedsynthetic peptides said composition comprising at least five isolatedsynthetic peptides of at least five proteins selected from LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, GSLG1,KIT or GGH wherein the at least five isolated synthetic peptides areselected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQ ID NO: 58),QLLLTADDR (SEQ ID NO: 60), ADHHATNGVVHLIDK (SEQ ID NO: 136),DILATNGVIHYIDELLIPDSAK (SEQ ID NO: 137), LTLLAPLNSVFK (SEQ ID NO: 139),SPYQLVLQHSR (SEQ ID NO: 140), ADDILASPPR (SEQ ID NO: 331), AVGLAGTFR(SEQ ID NO: 332), TEAPSATGQASSLLGGR (SEQ ID NO: 335), GGSTSYGTGSETESPR(SEQ ID NO 554), NSLFEYQK (SEQ ID NO: 556), TVIGPDGHK (SEQ ID NO: 557),VQHIQLLQK (SEQ ID NO: 558), ALFQDIK (SEQ ID NO: 593), DDVALEGVSHFFR (SEQID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER (SEQ ID NO:596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598),LYGSAGPPPTGEEDTAEK (SEQ ID NO: 663), TWNDPSVQQDIK (SEQ ID NO: 664),VYEGERPLTK (SEQ ID NO: 665), IIIQESALDYR (SEQ ID NO: 666), LDPALQDK (SEQID NO: 667), LIAQDYK (SEQ ID NO: 668), NDINILK (SEQ ID NO: 669),VAELSSDDFHLDR (SEQ ID NO: 670), ALPGTPVASSQPR (SEQ ID NO: 839),EVPLLQSLWLAHNEIR (SEQ ID NO: 840), LPGLPEGAFR (SEQ ID NO: 841),TVAAGALASLSHLK (SEQ ID NO: 843), ASHEEVEGLVEK (SEQ ID NO: 938),STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQID NO: 942), AALSGANVLTLIEK (SEQ ID NO: 958), TVLWPNGLSLDIPAGR (SEQ IDNO: 959), VFFTDYGQIPK (SEQ ID NO: 960), ADEGISFR (SEQ ID NO: 1321),ATAVMPDGQFK (SEQ ID NO: 1322), DISLSDYK (SEQ ID NO: 1323), GLFIIDDK (SEQID NO: 1324), IGHPAPNFK (SEQ ID NO: 1325), LVQAFQFTDK (SEQ ID NO: 1326),QGGLGPMNIPLVSDPK (SEQ ID NO: 1327), QITVNDLPVGR (SEQ ID NO: 1328),SVDETLR (SEQ ID NO: 1329), TIAQDYGVLK (SEQ ID NO: 1330),GGTLSTPQTGSENDALYEYLR (SEQ ID NO: 1588), LDTLAQEVALLK (SEQ ID NO: 1589),NWETEITAQPDGGK (SEQ ID NO: 1590), FQDLVDAVR (SEQ ID NO: 1641),FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643),GFLLLASLR (SEQ ID NO: 1644), YYIAASYVK (SEQ ID NO: 643), YVSELHLTR (SEQID NO: 878), or GTSQNDPNWVVR (SEQ ID NO: 1645).
 9. The composition ofclaim 8, wherein the at least five isolated synthetic peptides areselected from GFLLLASLR (SEQ ID NO: 1644), AVGLAGTFR (SEQ ID NO: 332),ALPGTPVASSQPR (SEQ ID NO: 839), LDTLAQEVALLK (SEQ ID NO: 1589),LGGPEAGLGEYLFER (SEQ ID NO: 596), TWNDPSVQQDIK (SEQ ID NO: 664),ALQASALK (SEQ ID NO: 58), LTLLAPLNSVFK (SEQ ID NO: 139), VEIFYR (SEQ IDNO: 941), TVLWPNGLSLDIPAGR (SEQ ID NO: 959), NSLFEYQK (SEQ ID NO: 556),QITVNDLPVGR (SEQ ID NO: 1328), YYIAASYVK (SEQ ID NO: 643), YVSELHLTR(SEQ ID NO: 878), and IIIQESALDYR (SEQ ID NO: 666).
 10. A compositioncomprising at least 5 transition ion pairs said composition comprisingat least one transition ion pair of at least 5 proteins selected fromALDOA, TSP1, FRIL, KIT, and GGH, wherein the at least one transition ionpair consists of a precursor ion with a corresponding m/z and a fragmention with a corresponding ion m/z, and wherein the transition ion pairsare selected from precursor ALQASALK (SEQ ID NO: 58) transition pair401.25-260.2, 401.25-331.2, 401.25-489.3, or 401.25-617.4; precursorGFLLLASLR (SEQ ID NO: 1644) transition pair 495.31-318.2, 495.31-375.2,495.31-446.3, or 495.31-559.4;precursor YYIAASYVK (SEQ ID NO: 643)transition pair 539.28-567.3, 539.28-638.40, 539.28-751.4, or539.28-914.5; precursor LGGPEAGLGEYLFER (SEQ ID NO: 596) transition pair804.41-451.2, 804.41-525.3, 804.41-564.3, or 804.41-913.4; and precursorYVSELHLTR (SEQ ID NO: 878) transition pair 373.21-263.1, 373.21-276.2,373.21-526.3, or 373.21-639.4, wherein each of the precursor ions has adouble or triple charge.
 11. The composition of claim 10, furthercomprising an additional five transition ion pairs comprising onetransition ion pair of at least one protein selected from LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT,GGH or GSLG1.
 12. The composition of claim 11, further comprising anadditional five transition ion pairs comprising one transition ion pairof at least one protein selected from APOE, BASP1, CD14, FOXA2 or HSPB1,wherein the additional one transition ion pair consists of a precursorion with a corresponding m/z and a fragment ion with a correspondingm/z, and wherein the transition ion pairs are selected from precursorAATVGSLAGQPLQER (SEQ ID NO: 82) transition pair 479.4-600.3,479.4-642.4, 479.4-827.4, or 479.4-898.5; precursor ALMDETMK (SEQ ID NO:83) transition pair 469.72-508.2, 469.72-469.72, 469.72-508.2,469.72-623.3, 469.72-754.3, or 469.72-867.4; precursor GEVQAMLGQSTEELR(SEQ ID NO: 85) transition pair 824.4-616.3, 824.4-729.4, 824.4-734.4,or 824.4-919.4; precursor LAVYQAGAR (SEQ ID NO: 86) transition pair474.77-502.3, 474.77-665.3, 474.77-764.4, or 474.77-835.4; precursorLGPLVEQGR (SEQ ID NO: 87) transition pair 484.78-588.3, 484.78-701.4,484.78-798.4, or 484.78-855.5; precursor SELEEQLTPVAEETR (SEQ ID NO: 88)transition pair 865.93-459.2, 865.93-801.4, 865.93-902.5, or865.93-930.4; precursor SWFEPLVEDMQR (SEQ ID NO: 89) transition pair768.86-274.1, 768.86-421.2, 768.86-678.3, or 768.86-987.5; precursorWELALGR (SEQ ID NO: 90) transition pair 422.74-316.1, 422.74-345.2,422.74-529.3, or 422.74-658.4; precursor AAEAAAAPAESAAPAAGEEPSK (SEQ IDNO: 118) transition pair 983.97-414.2, 983.97-485.2, 983.97-556.3, or983.97-885.4; precursor AEGAATEEEGTPK (SEQ ID NO: 119) transition pair645.3-258.1, 645.3-789.4, 645.3-890.4, or 645.3-961.4; precursorAPEQEQAAPGPAAGGEAPK (SEQ ID NO: 120) transition pair 592.62-629.3,592.62-754.3, 592.62-825.4, or 592.62-951.5; precursor EKPDQDAEGK (SEQID NO: 122) transition pair 558.76-404.2, 558.76-647.3, 558.76-859.4, or558.76-913.4; precursor ESEPQAAAEPAEAK (SEQ ID NO: 123) transition pair714.34-515.3, 714.34-644.3, 714.34-786.4, or 714.34-857.4; precursorETPAATEAPSSTPK (SEQ ID NO: 124) transition pair 693.84-616.3,693.84-816.4, 693.84-917.5, or 693.84-988.5; precursor GYNVNDEK (SEQ IDNO: 125) transition pair 469.71-276.2, 469.71-505.2, 469.71-604.3, or469.71-718.3; precursor SDGAPASDSKPGSSEAAPSSK (SEQ ID NO: 126)transition pair 644.96-260.1, 644.96-331.1, 644.96-418.2, or644.96-586.2; precursor TEAPAAPAAQETK (SEQ ID NO: 127) transition pair642.83-541.3, 642.83-744.4, 642.83-815.4, or 642.83-983.5; precursorAFPALTSLDLSDNPGLGER (SEQ ID NO: 206) transition pair 987-500.3,987-628.3, 987-742.4, or 987-944.4; precursor ATVNPSAPR (SEQ ID NO: 207)transition pair 456.75-386.2, 456.75-430.2, 456.75-527.3, or456.75-740.4; precursor ELTLEDLK (SEQ ID NO: 208) transition pair480.77-260.2, 480.77-504.3, 480.77-617.4, or 480.77-718.4; precursorFPAIQNLALR (SEQ ID NO: 209) transition pair 571.84-714.4, 571.84-827.5,571.84-898.5, or 571.84-995.6; precursor LTVGAAQVPAQLLVGALR (SEQ ID NO:211) transition pair 593.03-416.3, 593.03-515.3, 593.03-628.4, or593.03-740.4; precursor NVSWATGR (SEQ ID NO: 212) transition pair445.73-301.2, 445.73-404.2, 5, 445.73-590.3, or 445.73-677.3; precursorSTLSVGVSGTLVLLQGAR (SEQ ID NO: 213) transition pair 879.51-431.2,879.51-544.3, 879.51-657.4, or 879.51-970.6; precursor SWLAELQQWLKPGLK(SEQ ID NO: 214) transition pair 599.67-274.1, 599.67-414.3,599.67-542.4, or 599.67-841.5; precursor VDADADPR (SEQ ID NO: 215)transition pair 429.7-272.2, 429.7-573.3, 429.7-644.3, or 429.7-759.3;precursor AYEQVMHYPGYGSPMPGSLAMGPVTNK (SEQ ID NO: 577) transition pair961.45-558.3, 961.45-615.3, 961.45-817.4, or 961.45-930.5; precursorEAAGAAGSGK (SEQ ID NO: 578) transition pair 409.7-348.2, 409.7-490.3,409.7-547.3, or 409.7-618.3; precursor MHSASSMLGAVK (SEQ ID NO: 579)transition pair 406.87-374.2, 406.87-487.3, 406.87-601.2, or406.87-732.3; precursor TGLDASPLAADTSYYQGVYSRPIMNSS (SEQ ID NO: 582)transition pair 955.45-387.2, 955.45-545.3, 955.45-648.3, or955.45-826.4; precursor AQLGGPEAAK (SEQ ID NO: 731) transition pair471.26-313.2, 471.26-427.2, 471.26-629.3, or 471.26-742.4; precursorDGVVEITGK (SEQ ID NO: 732) transition pair 459.25-305.2, 459.25-418.3,459.25-547.3, or 459.25-646.4; precursor GPSWDPFR (SEQ ID NO: 733)transition pair 481.23-419.2, 481.23-534.3, 481.23-720.3, or481.23-807.4; precursor LATQSNEITIPVTFESR (SEQ ID NO: 734) transitionpair 953.5-639.3, 953.5-744.4, 953.5-835.4, or 953.5-857.4; precursorLFDQAFGLPR (SEQ ID NO: 735) transition pair 582.31-272.2; 582.31-442.3,582.31-660.4, or 582.31-903.5; precursor QDEHGYISR (SEQ ID NO: 736)transition pair 368.84-262.2, 368.84-538.3, 368.84-595.3, 368.84-730.3,368.84-504.3, 368.84-660.4, 368.84-747.4, or 368.84-834.4; precursorVPFSLLR (SEQ ID NO: 738) transition pair 416.26-488.3, 416.26-488.3,416.26-635.4, or 416.26-732.4; or precursor VSLDVNHFAPDELTVK (SEQ ID NO:739) transition pair 595.31-347.2, 595.31-460.3, 595.31-801.4, or595.31-872.5, wherein each of the precursor ions has a double or triplecharge.
 13. The composition of claim 11, wherein the at least fivetransition ion pairs are selected from precursor GFLLLASLR (SEQ ID NO:1644) transition pair 495.31-559.40, precursor LGGPEAGLGEYLFER (SEQ IDNO: 596) transition pair 804.40-913.40, precursor ALQASALK (SEQ ID NO:58) transition pair 401.25-617.40, YYIAASYVK (SEQ ID NO: 643) transitionpair 539.28-638.40, precursor LGGPEAGLGEYLFER (SEQ ID NO: 596)transition pair 804.41-451.2, 804.41-525.3, 804.41-564.3, 804.41-913.4,and precursor YVSELHLTR (SEQ ID NO: 878) transition pair 373.21-263.1,373.21-276.2, 373.21-526.3, 373.21-639.4.
 14. A method comprisinganalyzing the composition of claim 8 using mass spectrometry.
 15. Themethod of claim 14, wherein the method uses selected reaction monitoringmass spectrometry.
 16. A method comprising analyzing the composition ofclaim 8 using Enzyme-Linked Immunosorbent Assay (ELISA).
 17. Amultiplexed LC-SRM-MS assay for the measurement of a plurality ofproteins in a single sample comprising: a) generating a set of peptidesand corresponding transitions for each protein to be monitored; b)optimizing the collision energy for each transition such thatinterference among the transitions is avoided; c) selecting a set oftransitions that have the greatest peak areas for each protein, andwherein the selected transitions do not interfere with the ions in thesample; d) measuring a plurality of proteins in a sample.
 18. The assayaccording to claim 17 wherein the two best peptides per protein and thetwo best transitions per peptide are selected based on experimental dataresulting from LC-SRM-MS analysis of one or more of the followingexperimental samples: a biological disease sample, a biological controlsample, and a mixture of synthetic peptides.
 19. The assay according toclaim 18 wherein the biological disease and biological control samplesare processed using an immunodepletion method prior to LC-SRM-MSanalysis.
 20. The assay according to claim 19, wherein the experimentalsamples contain internal standard peptides.
 21. The assay according toclaim 19, wherein the LC-SRM-MS analysis method specifies a maximum of7000 transitions, including transitions of the internal standardpeptides and transitions.
 22. The Assay according to claim 17, whereinthe top two transitions per peptide are selected according to one ormore of the following criteria: (1) the transitions exhibit the largestpeak areas measured in either of the two biological samples; (2) thetransitions are not interfered with by other ions; (3) the transitionsdo not exhibit an elution profile that visually differs from those ofother transitions of the same peptide; (4) the transitions are notbeyond the detection limit of both of the two biological experimentalsamples; and (5) the transitions do not exhibit interferences.
 23. Theassay according to claim 17, wherein the top two peptides per proteinare selected according to one or more of the following criteria: (1) oneor more peptides exhibit two transitions according to claim 20 andrepresent the largest combined peak areas of the two transitionsaccording to claim 20; and (2) one or more peptides exhibit onetransition according to claim 20 and represent the largest combined peakareas of the two transitions according to claim
 20. 24. An assaydeveloped according to claim 17.